CN102781952A - Nucleic acid molecules encoding rantes, and compositions comprising and methods of using the same - Google Patents

Abstract

Nucleic acid molecules comprising a nucleotide sequence encoding RANTES and fragments and variants thereof are disclosed. Additionally, nucleic acid molecules and compositions comprising the nucleotide sequence encoding RANTES and fragments and variants thereof in combination with nucleic acid sequences encoding immunogens are provided. Recombinant viral vectors comprising the nucleotide sequence encoding RANTES and fragments and variants thereof with or without a nucleic acid sequence encoding immunogens are also provided as are live attenuated pathogens comprising a nucleotide sequence encoding RANTES and fragments and variants thereof. Methods of modulating immune responses and of inducing an immune response against an immunogen are also disclosed.

Description

The nucleic acid molecule of coding RANTES, the compsn that comprises it with and method of use

Invention field

The nucleic acid molecule of RANTES the present invention relates to encode.The present invention relates to comprise the vaccine of nucleotide sequence of RANTES of encoding, and preventative and/or therapeutic immunization is individual resisting immunogenic method, also to relate to the immunotherapeutic composition of the nucleotide sequence that comprises the RANTES that encodes, and immunotherapy method.

Background of invention

The application requires the right of priority of No. the 61/302nd, 324, U.S. Provisional Application, and it incorporates this paper into way of reference integral body.

RANTES; T cell expressing that it is regulated during for activation, normal and excretory (Regulated upon Activation; Normal T-cell Expressed, and Secreted) abbreviation is the 8kDa protein that is classified as chemoattracting cytoking or chemokine.RANTES is accredited as the gene of expressing in several days behind the T cell activation.Found the ACC chemokine of in many human diseasess, expressing, RANTES is regulated by Kruppel like factor 13 (KLF13) in the T lymphocyte.

With the interactional RANTES of Chemokine Receptors CCR3, CCR5 and CCR1 T cell, eosinophilic granulocyte and basophilic granulocyte had chemotaxis.It participates in white corpuscle raising to inflammatory site.The propagation and the activation of RANTES and some NK of zygotic induction (NK) cell of cytokine IL-2 that discharges by the T cell and IFN-γ.Such cell is known as CHAK (CC-chemokine activated kills and wounds) cell.

The U. S. application of incorporating this paper with way of reference into discloses compsn and the method for the nucleic acid molecule of use coding RANTES as immunotherapeutic agent and vaccine component for the 09/622nd, No. 452.When the expression of RANTES when the immunotherapeutic agent has changed in the individuality of expressing it immune aspect some.Similarly, the expression of RANTES has strengthened immunoreactive some aspect of antagonism vaccine immunogens when as vaccine a part of.

Immunotherapy is meant that mediator's immunoreation is to give desired therapeutic action.Immunotherapeutic agent is meant that when being applied to individuality, regulating individual immunity system is enough to finally reduce the symptom relevant with the immunoreation of not expecting or be enough to through strengthening those compsns that desired immunoreation comes final mitigation symptoms.In some situation; Immunotherapy is the part of vaccine inoculation rules; In said vaccine inoculation rules, individuality is used said vaccine; This vaccine is exposed in the case individuality and individually produces immunoreactive immunogen for resisting it, and reaction of immunotherapeutic agent enhancing immunity and/or selectivity strengthen treatment or prevention particular condition, infection or the required immunoreactive part (for example cellular immunization (cellular arm) or humoral immunization (humoral arm)) of disease.

The vaccine rules can be improved with the immunoreactive reagent of inducing improvement through sending mediator's immunoreation.Individuality is used make individuality be exposed to individual antagonism it produces in some inoculation rules of immunoreactive immunogenic vaccine, provide enhancing immunity reaction and/or selectivity to strengthen reagent as far as the required immunoreactive part (for example cellular immunization or humoral immunization) of treatment or prevention particular condition, infection or disease.

Vaccine for immune body with antagonism target antigen for example allergen, pathogen antigen or be useful with the relevant antigen of cell of participating in human diseases.Comprise tumour antigen that cancer is relevant and the antigen of being correlated with the relevant antigen of cell of participating in human diseases with the cell of participating in autoimmune disease.

When designing such vaccine, have recognized that the vaccine that in the individual cell of vaccination, produces target antigen is effective in the cellular immunization of induction of immunity system.Particularly, cause antigenic generation in the attenuated live vaccine of use non-toxic carrier and dna vaccination, the cell of each comfortable vaccinated individuality of recombiant vaccine, this causes inducing immune cellular immunization.On the other hand, although killed vaccine or inactivated vaccine and only comprise proteinic subunit vaccine and can induce effective humoral response are not induced the good cell immunoreation.

Cell immune response is for the protection that the enantiopathy pathogen infection is provided and normally necessary for the effective immune-mediated treatment that is provided for treating pathogenic infection, cancer or autoimmune disease.Therefore, the vaccine that in the cell of vaccinated individuality, produces target antigen for example uses attenuated live vaccine, recombiant vaccine and the dna vaccination of non-toxic carrier normally preferred.

Studied direct administration of nucleic acid sequence send protein or to animal and human's disease carry out vaccine inoculation and make great efforts in a large number to concentrate on delivery of nucleic acids effectively with efficient means so that produce treatment/adjuvant protein and/or expect antigenic necessity expression.

Dna vaccination has many advantages that are superior to more traditional gene delivery and method of vaccination (for example deactivation virus and recombinant protein class vaccine).Dna vaccination safety, stablize, be easy to produce; And well tolerable in the mankind; Wherein preclinical test shows the less [Martin of plasmid integration sign; T. etc., Plasmid DNA malaria vaccine:the potential for genomic integration after intramuscular injection.Hum Gene Ther, 1999.10 (5): the 759-68 page or leaf; Nichols, W.W. etc., Potential DNA vaccine integration into host cell genome.Ann N Y Acad Sci, 1995.772: the 30-9 pages or leaves].In addition; Because the antibody titers that the effectiveness of vaccine is not pre-existing in is to the influence of carrier, dna vaccination is very suitable for repeated administration [Chattergoon, M.; J.Boyer; And D.B.Weiner, Genetic immunization:a new era in vaccines and immune therapeutics.FASEB J, 1997.11 (10): the 753-63 page or leaf].Yet; A major obstacle of clinical employing dna vaccination is the immunogenic reduction [Liu of platform (platform) when transferring to than large animal; M.A. and J.B.Ulmer, Human clinical trials ofplasmid DNA vaccines.Adv Genet, 2005.55: the 25-40 pages or leaves].The state-of-the-art technology progress of dna vaccination immunogen engineering; Optimize and the interpolation of Tegeline leader sequence has improved the expression and the immunogenicity [Andre of dna vaccination like codon optimized, RNA; S.; Deng, Increased immune response elicited by DNA vaccination with a synthetic gp 120sequence with optimized codon usage.J Virol, 1998.72 (2): the 1497-503 page or leaf; Deml; L.; Deng; Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1Gag protein.J Virol, 2001.75 (22): the 10991-1001 page or leaf; Laddy, D.J., etc., Immunogenicity of novel consensus-based DNA vaccines against avian influenza.Vaccine, 2007.25 (16): the 2984-9 page or leaf; Frelin; L. etc., Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis Cvirus nonstructural 3/4A gene.Gene Ther, 2004.11 (6): the 522-33 page or leaf]; And the technology of developing recently in the plasmid delivery system; As electroporation [Hirao, L.A., etc.; Intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques.Vaccine, 2008.26 (3): the 440-8 page or leaf; Luckay; A. etc.; Effect ofplasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques.J Virol, 2007.81 (10): the 5257-69 page or leaf; Ahlen; G.; Deng, In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression; Inflammation, and infiltration of CD3 ⁺T cells.J Immunol, 2007.179 (7): the 4741-53 page or leaf].In addition; Research has shown uses total immunogen (consensus immunogen) to compare with independent natural antigen; Can strengthen the width [Yan of cell immune response; J. etc., Enhanced cellular immune responses elicited by an engineered HIV-1subtype B consensus-based envelope DNA vaccine.Mol Ther, 2007.15 (2): the 411-21 page or leaf; Rolland, M. etc., Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1proteins.J Virol, 2007.81 (16): the 8507-14 page or leaf].

Be used for the nucleic acid delivery sequence for example a kind of method of DNA be electroporation (EP) technology.This technology has been used to people's clinical trial to send anticancer disease drug, for example NSC 125066 and be used for many preclinical studies of a large amount of animal species.

Though vaccine is normally effective with enantiopathy pathogen infection or human diseases for preventative or therapeutic ground immune body, still deposits the demand to the vaccine with improvement.Need to produce immunoreactive compsn of enhanced and method.Likewise, though some immunotherapeutic agents are useful for the immunoreation of regulating in the patient, but still exist the immunotherapeutic composition with improvement and the demand of method.

Summary of the invention

The present invention relates to comprise the nucleic acid molecule that is selected from by the nucleotide sequence of the following group of forming: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide.

The invention still further relates to compsn; Said compsn comprises a plurality of one or more nucleic acid molecule, and it comprises one or more nucleotide sequences that are selected from by the following group of forming: 1) be selected from the group of being made up of following sequence: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ IDNO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide; And b) one or more immunogenic one or more other nucleotide sequences of encoding.

The present invention relates to the immunoreactive method of regulating in addition; Said method comprises that said nucleic acid molecule comprises the nucleotide sequence that is selected from by the following group of forming to the step of individual administration of nucleic acid molecule or compsn: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ IDNO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide; Said compsn comprises a plurality of one or more nucleic acid molecule, and it comprises one or more nucleotide sequences that are selected from by the following group of forming: 1) be selected from the group of being made up of following: SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide; And b) one or more immunogenic one or more other nucleotide sequences of encoding.

The invention further relates to and comprise the recombinant viral vector that is selected from by the nucleotide sequence of the following group of forming: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ IDNO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ IDNO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide.

The invention still further relates to the intraindividual immunoreactive method of regulating; Said method comprises that said recombinant viral vector comprises the nucleotide sequence that is selected from by the following group of forming to said individual administered recombinant virus vector: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide.

The present invention relates in addition and comprises the deactivation pathogenic agent that is selected from by the nucleotide sequence of the following group of forming: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide.

The invention still further relates to the method for immune body with the enantiopathy substance; Said method comprises to said individuality uses the deactivation pathogenic agent, and said deactivation pathogenic agent comprises the nucleotide sequence that is selected from by the following group of forming: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ IDNO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide.

The accompanying drawing summary

Fig. 1 a-1e illustrates and is illustrated in the inductive data of carrying out the strong cell immune response in DNA inoculation back with EP.Each immunity back is shown as stack group average response ± SEM to the total reaction of SIVgag (white band), env (fascia cinerea) and pol (black band).Use Du Ke to check (Tukey post hoc test) and Deng Nite T3 to check (Dunnett T3post hoc) to come to measure respectively the significance of difference between the group of third and fourth immunity afterwards afterwards.

Fig. 2 a-2c illustrates the multiplication capacity of exsomatize behind the dna immunization (ex-vivo).To in vitro stimulate 5 days to measure the multiplication capacity of antigen-specific sexual cell with CFSE dyeing and with SIVgag (informal voucher band), env (fascia cinerea) and pol (black band) peptide at the 4th isolating fresh PBMC of two week of immunity back.Among Fig. 2 a, show the point diagram of representative animal.Total SIV proliferative response is organized average response ± SEM for CD4 as stack ⁺The T cellular compartment is shown in Fig. 2 b and for CD8 ⁺The T cellular compartment is shown among Fig. 2 c.Graceful through matching-Whitney check (pair-wise Mann-Whitney test), proofread and correct the significant difference of measuring between each group with Bang Feiluoni (Bonferroni), < 0.017 is remarkable to the p value.

Fig. 3 a-3d illustrates the multi-functional collection of illustrative plates of immunity back SIVpol reaction.Use SIVpol peptide storehouse mixture (peptide pool mix) to stimulate in vitro 5 hours at the 4th immunity back 2 all isolating PBMC.Pair cell dyeing is with the generation of IFN γ, TNF α and IL-2 in the analysis of cells and the threshing through CD107a.Fig. 3 a illustrates for the painted representativeness of the cell within a cell factor and establishes an analysis (gating analysis).Distinguish singlet colony (singlet population) through using forward scatter height (FSC-H) and forward scatter area (FSC-A).We use lateral scattering area (SSC-A) to come isolated lymphocytes colony through FSC-A subsequently.The T cell of living is removed to establish for the Pacific Blue to viability, CD14, CD16 and CD19 and is accredited as negative staining (dump gate) (comprising light violet look dyestuff (Violet Vivid Dye)), and for the positive dyeing of CD3.After this, at CD8 with respect to IFN γ ⁺And CD4 ⁺Establish an incident proof downward modulation on the incident continuously.Be depicted as in this embodiment and identify CD8 ⁺The T cell establish door.The one CD3 ⁺CD8 ⁺The T cell is through positive CD8 dyeing and negative CD4 dyeing, can have any CD4 of the CD8 of rise after the eliminating activation ⁺The T cell is identified.Natural CD8 is got rid of in CD28 and CD95 dyeing from analyze ⁺The T cell.Establish a gained in four functions among the group at us: CD107a, IL-2, IFN γ and the TNF α each then and experience antigenic CD8 ⁺The T cell.Bar chart representing among Fig. 3 a and the 3b frequency of each in 15 kinds of function combinations.Fig. 3 b illustrates CD4 ⁺T cell responses.Fig. 3 c illustrates CD8 ⁺T cell responses.Pie chart illustrates the SIVpol specific C D4 of have 4 kinds of functions (purple or grey black look), 3 kinds of functions (yellow or light ash), 2 kinds of functions (green or medium grey black look) or a kind of function (light blue or grey) ⁺The ratio of T cell.Be superimposed on the sum frequency of the numeral SIVpol reaction on the pie chart.Fig. 3 d relates to IFN γ ⁺Single function CD8 ⁺The memory phenotype of T cell.IFN γ ⁺The point diagram of single function (grey black look) is superimposed on CD28 through the CD95 density map and goes up to measure the memory phenotype of this colony.Be illustrated in the dyeing in the DNA+12 group from representative animal.

Fig. 4 a and 4b illustrate keeping of multipurpose memory T cell colony.In the end immunity (day that SIVmac251 excites) back eight months isolating PBMC in vitro stimulated 5 hours with SIVpol peptide storehouse mixture.Pair cell dyeing is to produce IFN γ, TNF α and IL-2 and to pass through the CD107a threshing in cell.Bar chart representing among Fig. 4 a and the 4b frequency of each in 15 kinds of function combinations.Fig. 4 a illustrates CD4 ⁺T cell responses.Fig. 4 b illustrates CD8 ⁺T cell responses.Pie chart illustrates the SIVpol specific C D4 of have 4 kinds of functions (purple or grey black look), 3 kinds of functions (yellow or light gray), 2 kinds of functions (green or medium grey black look) or a kind of function (light blue or grey) ⁺The ratio of T cell.The numeral SIVpol reaction that is superimposed on the pie chart is frequently tired.

Fig. 5 a-5d illustrates the data that excite from the SIVmac251 mucous membrane.Fig. 5 a be illustrated in excite preceding, inoculate (or light gray) when exciting back the 2nd week (peak), the 14th week (a setting point) and do not inoculate the comparison of the virus load between the animal of (● or grey black look) with the 35th week (for a long time).Fig. 5 b illustrates through group: exciting of natural (● or grey black look), DNA (● or grey), DNA+12 (● or medium grey black look), DNA+RANTES ( or light gray) is preceding, the comparison of peak, a setting point and long-term virus load.Fig. 5 c illustrates the area under the graphic representation.Cell mean is shown.Fig. 5 d shows protectiveness I class allelotrope, Mamu-B*03 and Mamu-B*017 ( or light gray) and the comparison of the virus load between non-contrast allelotrope (■ or the grey black look) animal between peak, a setting point and long-term period of infection.Carry out two tail T checks with the significance of mensuration, and use has the ANOVA that two tail Deng Nite check (Dunnett post hoc test) afterwards in Fig. 5 b for the difference between the group of Fig. 5 a and 5d.

Fig. 6 a-6e is illustrated in the CD4 after the SIVmac251 mucous membrane excites ⁺The T cell depletion.Be illustrated in and excite back periphery CD4 ⁺The Cytometric variation of T.CD4 ⁺The Cytometric relative variation of T is described as the per-cent of baseline counts and for natural (■), DNA (▲), DNA+12

And DNA+RANTES (

) organize to illustrate and organize average ± SEM.Excite the indivedual CD4 in back ⁺The time course of T cell depletion.The outstanding demonstration has and the animal of protecting relevant haplotype in each group.Except in having the allelic DNA+12 of Mamu-B*017 group 4394 all have Mamu-B*003 allelotrope.

Fig. 7 a and 7b illustrate immunity back CCR5 ⁺Inducing of T cell.PBMC in immunity (that is, being used for the last immunity of inducing molecule adjuvant) isolating freezing preservation in back for the third time stimulates with the SIVpol peptide, and through identifying the antigen-specific sexual cell by the generation of IFN γ, TNF α, IL-2 or the CD107a of ICS mobilization.Mensuration is for CD8 ⁺(data are shown in Fig. 7 a) and CD4 to the T cellular compartment ⁺The SIVpol specificity of T cellular compartment (data are shown in Fig. 7 b), CCR5 ⁺The frequency of cell.Data presentation is a frequency in total CD8 or CD4 colony.IFN γ is attached on the identical fluorophor with TNF α, in this group, to adapt to other dyeing.

Describe in detail

The nucleic acid molecule of coding human RANTES is provided, and it has the nucleotide sequence (SEQ ID NO:1) that is designed in people's cell, produce high expression level.Aminoacid sequence by nucleotide sequence coded RANTES is shown among the SEQ ID NO:2.Nucleotide sequence can be operatively attached to the required controlling element of expression in people's cell.Can provide other element and sequence with further enhancing expression level.Plasmid, virus vector or the cell that comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 can be applied to individuality and in the cell of individuality, express, with function RANTES protein delivery to individual.In addition, can be to cultivating with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 transformed host cells to produce RANTES protein.

When sending as the part of immunotherapeutic agent or vaccine, the nucleic acid molecule of coding RANTES and function fragment thereof is regulated immunoreation.Therefore, the nucleic acid molecule of coding RANTES and function fragment thereof can be used as immunotherapeutic agent and/or sends with the vaccine combination or as components in vaccines, and said vaccine component comprises that also coding needs the nucleic acid molecule of the immunogenicity target of immunoreation antagonism.

Though not fettered by scientific theory, be used to regulate immunoreactive immunotherapeutic agent and can comprise nucleic acid molecule, said nucleic acid molecule comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.Can be used for exciting the immunoreactive vaccine of the immunogenic enhanced of antagonism can comprise in following one or more: the nucleic acid molecule that 1) comprises SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5 or SEQ ID NO:7 and the immunogenic nucleotide sequence of coding target; 2) comprise first nucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 and comprise second nucleic acid molecule of coding target immunogenic nucleotide sequence.

Can use such nucleic acid molecule to carry out immunotherapy and immunization method.In order to realize the high level expression of RANTES, can be to have the controlling element that is operably connected to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 with immunotherapeutic agent and vaccine production.

The nucleotide sequence of coding RANTES or its function fragment is delivered to individuality regulates intraindividual immunoreation.When sending as immunotherapeutic agent, the nucleotide sequence of coding RANTES or its function fragment is regulated intraindividual immunoreation relates to immune various diseases, the patient's condition and illness with alleviation the symptom or the cause of disease.When being delivered to individuality with the immunogenic nucleotide sequence of coding, strengthen the immunogenic immunoreation of antagonism as components in vaccines.When the nucleic acid molecule of coding RANTES or its function fragment was applied to individuality, RANTES was absorbed by cell and is expressed in the cell, therefore, the RANTES protein delivery was arrived individual.With regard to vaccine, also use the immunogenic nucleotide sequence of coding, it is absorbed by cell and expresses, and produces immunogenic protein thus.The method of sending protein coding sequence can comprise sends a plurality of mononucleotide molecules or a plurality of multiple different nucleic acid molecules.Proteinic encoding sequence can be a part, recombiant vaccine or the attenuated vaccine of for example plasmid.

Provide individuality is carried out preventative and/or therapeutic immunization compsn and the method with the cell of enantiopathy substance or unusual, disease-related.Said vaccine can be any vaccine type such as attenuated live vaccine, recombiant vaccine or nucleic acid or dna vaccination.Through sending the nucleic acid molecule of coding immunogen and RANTES or its function fragment, can regulate by vaccine-induced immunoreation.When via effable nucleic acid molecule, RANTES is useful especially when for example sending as the part of plasmid or recombinant vectors or deactivation pathogenic agent or cell.When not infecting or do not have in the disease individuality in order to induce protective immunological reaction preventative sending, RANTES is useful especially.The nucleic acid molecule of coding RANTES can be sent in not celliferous compsn.In some embodiments, can use the nucleic acid molecule of coding RANTES and not have any other cytokine.

Use standard technique and the raw material that is easy to obtain to prepare the proteinic nucleic acid molecule of coding RANTES.

Be provided for sending RANTES compsn, use said method for compositions, and vaccine and immunization method.Said compsn comprises nucleic acid molecule, and said nucleic acid molecule comprises the coding RANTES that is operably connected to controlling element or the nucleotide sequence of its function fragment.When being vaccine a part of, said compsn comprises the immunogenic nucleotide sequence of the coding that is operably connected to controlling element.Can the Injectable composition that comprise said composition be applied to individuality.

1. definition

The term that this paper uses has been merely the description particular, is not to be intended to limit.Singulative " ", " a kind of " and " being somebody's turn to do " as using in specification sheets and the appended claims comprise a plurality of indicators only if context is clearly stipulated.

For quoting of this paper numerical range, comprise intervenient number clearly with same accuracy.For example,, except 6 and 9, also comprise several 7 and 8,, comprise numeral 6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9 and 7.0 clearly for the scope of 6.0-7.0 for the scope of 6-9.

A. adjuvant

" adjuvant " that uses like this paper refers to add to DNA plasmid vaccine as herein described to strengthen any molecule of the immunogenicity of antigens of being encoded by hereinafter described DNA plasmid and nucleic acid sequence encoding.

B. antibody

" antibody " that uses like this paper refers to IgG, IgM, IgA, IgD or IgE antibody-like, and perhaps its fragment, fragment or verivate comprise Fab, F (ab') 2, Fd and single-chain antibody, duplex, bi-specific antibody, bifunctional antibody and verivate thereof.Said antibody can be the mixture that shows these antibody of enough binding specificities from the antibody of mammiferous serum sample isolated antibody, polyclonal antibody, affinity purification or to the epi-position of expectation or by its deutero-sequence.

C. encoding sequence

" encoding sequence " or " coding nucleic acid " that uses like this paper refers to comprise the nucleic acid (RNA or dna molecular) of the nucleotide sequence of coded protein.Encoding sequence can further comprise exercisable start signal and the termination signal that is connected to regulatory element, comprises the promotor and the polyadenylation signal that can instruct at the individual or mammiferous cell inner expression of using said nucleic acid.

D. complementary

" complementary (complement) " or " complementary (complementary) " that uses like this paper refers to that nucleic acid can form Wo Sen-Ke Like (Watson-Crick) (for example, A-T/U and C-G) or Hu Sitan (Hoogsteen) base pairing between the Nucleotide of nucleic acid molecule or nucleotide analog.

E. constant current

" constant current " that use like this paper refer to during electricimpulse is sent to homologue, by said tissue or define the electric current that said cells of tissues is accepted or experienced.Electricimpulse is transmitted by electroporation device as herein described.Because the electroporation device that provides of this paper has feedback element, preferably has instantaneous feedback, thus electricimpulse in the cycle this electric current in said tissue, remain in constant amperes.Feedback element can be measured the resistance that runs through impulse duration tissue (or cell), and causes that electroporation device changes its electric energy output (for example, increasing voltage) and therefore runs through electricimpulse (about several microseconds) and keep constant from the electric current of pulse to pulse in homologue.In some embodiments, feedback element comprises unit.

F. current feedback or feedback

" current feedback " or " feedback " commutative use, and be meant the active response of the electroporation device that provides, it comprise the in-house electric current between the potential electrode and correspondingly change the energy output that transmits by EP equipment so as holding current in constant level.This constant level was preset before causing pulse sequence or electrical treating by the user.Feedback can be attended by the electroporation assembly of electroporation device; Unit for example because circuit wherein continuously the in-house electric current between the monitoring electrode and relatively monitoring electric current (or in-house electric current) and preset electric current and energy output is regulated with the electric current that keeps monitoring in preset level.Because it is the analog closed-loop feedback, so feedback loop can be moment.

G. scattered current

" scattered current " that this paper uses refers to from the current-mode of the various pin electrode arrays transmission of electroporation device as herein described; Wherein said pattern minimizes, or preferably eliminates the generation of the relevant thermal stresses of electroporation on any zone of the tissue of treating electroporation.

H. electroporation

" electroporation ", " electricity passes through and changes " or " electrokinetic force enhancing " (" EP ") like the commutative use of this paper refer to use the transmembrane electric field pulse to induce the microcosmic path (hole) in the microbial film; Their existence allows biomolecules, and for example plasmid, oligonucleotide, siRNA, medicine, ion and water pass through to opposite side from a side of cytolemma.

I. Feedback mechanism

" Feedback mechanism " that uses like this paper refers to the processing carried out through software or hardware (or firmware); This handle to accept and with required tissue (energy pulse transmit before, during and/or afterwards) impedance and preset value; Preferred electric current compares, and the energy pulse that adjusting transmits is to reach preset value.Can carry out Feedback mechanism through the simulation closed loop circuit.

J. function fragment

" function fragment " that use like this paper is meant following nucleic acid molecule for SEQ ID NO:1: 1) have and nucleic acid molecule less than a part of corresponding nucleotide sequences (promptly except that its total length SEQ ID NO:1) of the SEQ ID NO:1 of total length SEQ ID NO:1; With 2) coding has the nucleic acid molecule of the polypeptide of RANTES activity (promptly when in the mankind, express, its RANTES as generation when SEQ ID NO:1 expresses plays a role) in the mankind.The size of function fragment can be 90 or above, 120 or above, 150 or above, 180 or above, 210 or above or 240 or above length.Dna fragmentation can be less than 10 Nucleotide, less than 20, less than 30, less than 40, less than 50, less than 60, less than 75, less than 90, less than 120, less than 150, less than 180, less than 210 or less than 240.For the disclosed purpose of this paper, expect size as herein described also constitute the size scope for example the size of DNA function fragment can be the length of 20-90,20-120,20-150,20-180,20-210,20-240,30-90,30-120,30-150,30-180,30-210,30-240,40-90,40-120,40-150,40-180,40-210,40-240,50-90,50-120,50-150,50-180,50-210,50-240,60-90,60-120,60-150,60-180,60-210,60-240,75-90,75-120,75-150,75-180,75-210,75-240,90-120,90-150,90-180,90-210,90-240,20-90,120-150,120-180,120-210,120-240,150-180,150-210,150-240,180-210,180-240 and 210-240.

" function fragment " refers to by as non-total length SEQ ID NO:2, the i.e. polypeptide of the function fragment nucleic acid molecule encoding except that SEQ ID NO:2 for peptide sequence.Polypeptide fragment can be 30 or above amino acid length, 35 or above, 40 or above, 45 or above, 50 or above, 55 or above, 60 or above, 65 or above, 70 or above, 75 or above, 80 or above, 85 or above or 90 or above length.Polypeptide fragment can be less than 10 amino acid, less than 15, less than 20, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, less than 70, less than 75, less than 80, less than 85 or less than 90 amino acid whose length.For in the disclosed purpose of this paper; Expect that size as herein described also constitutes the scope of size, for example the segmental size of protein function can be the length of 30-35,30-40,30-45,30-50,30-55,30-60,30-65,30-70,30-75,30-80,30-85,30-90,35-40,35-45,35-50,35-55,35-60,35-65,35-70,35-75,35-80,35-85,35-90,40-45,40-50,40-55,40-60,40-65,40-70,40-75,40-80,40-85,40-90,45-50,45-55,45-60,45-65,45-70,45-75,45-80,45-85,45-90,50-55,50-60,50-65,50-70,50-75,50-80,50-85,50-90,55-60,55-65,55-70,55-75,55-80,55-85,55-90,60-65,60-70,60-75,60-80,60-85,60-90,65-70,65-75,65-80,65-85,65-90,70-75,70-80,70-85,70-90,75-80,75-85,75-90,80-85,85-90 and 85-90.

K. genetic constructs

Be meant the DNA or the RNA molecule of the nucleotide sequence that comprises coded protein like the term " genetic constructs " of this paper use.Encoding sequence comprises the initial sum termination signal that is operably connected to controlling element, and said controlling element comprises can instruct expression promoter and polyadenylation signal in the cell of the individuality of administration of nucleic acid molecule.The term " but expression-form " that uses like this paper is meant the gene construct of the necessary controlling element that comprises the encoding sequence that is operably connected to coded protein, thereby when in the cell that is present in individuality, will express encoding sequence.

L. same

" same " or " identity " as in the context of two or more nucleic acid of this paper or peptide sequence, using refers to that said sequence has the residue of particular percentile identical in the specific region.Said per-cent can calculate through following: the quantity of the position that optimize two sequences of comparison, compare two sequences in the specific region, the same residue of mensuration occurs in two sequences is to produce the quantity of matched position; Quantity with matched position multiply by 100 to obtain the per-cent of sequence identity divided by the sum of position in the specific region with the result.Have the specific region that different lengths or comparison produce one or more staggered ends and comparison two sequences and only comprise in the situation of simple sequence, the simple sequence residue be contained in calculating denominator but not in the molecule.When comparison dna and RNA, can thymus pyrimidine (T) and uridylic (U) be regarded as quite.Identity can be artificial or through using the computer sequence algorithm for example BLAST or BLAST2.0 carry out.

M. impedance

When discussing Feedback mechanism, can use " impedance " and can convert current value into, therefore, can compare with scheduled current according to Ohm's law.

N. immunoreation

" immunoreation " used like this paper refers to host immune system, like mammiferous immune system response in the for example total antigen of influenza hemagglutinin and activation of antigen.Immunoreation can be the form of cell or humoral response, or both.

O. nucleic acid

" nucleic acid " or " oligonucleotide " or " polynucleotide " that uses like this paper refers to covalently bound at least two Nucleotide that arrive together.The description of strand has also defined the sequence of complementary strand.Therefore, nucleic acid also comprises the complementary strand of said strand.Can many variants of nucleic acid be used as given nucleic acid from identical purpose.Therefore, nucleic acid also comprises same basically nucleic acid and complementary strand thereof.Strand be provided under the stringent hybridization condition can with the probe of target sequence hybridization.Therefore, nucleic acid also is included in the probe of hybridization under the stringent hybridization condition.

Nucleic acid can be strand or two strands, perhaps can comprise the part of double-stranded and single stranded sequence.Nucleic acid can be DNA, genome and cDNA, RNA or hybrid; Its amplifying nucleic acid can comprise the combination of ribodesose and ribonucleotide and comprise the combination of the base of uridylic, VITAMIN B4, thymus pyrimidine, cytosine(Cyt), guanine, inosine, xanthine, xanthoglobulin, iso-cytosine and isoguanine.Nucleic acid can obtain through chemical synthesis or through recombination method.

P. be operably connected

" being operably connected " of using like this paper refers to that expression of gene is under the control of the promotor of space connection.Promotor can be located at the 5' (upper reaches) or the 3' (downstream) of the gene under its control.Distance between promotor and the gene can and promotor therefrom in the deutero-gene distance between the gene of promotor and its control roughly the same.As known in the art, can under the situation of not losing promoter function, regulate the variation of this distance.

Q. promotor

" promotor " used like this paper refers to give, activation or strengthen the molecule of the synthetic or natural origin of the expression of nucleic acid in cell.Promotor can comprise one or more concrete transcriptional regulatory sequences with its expression of further enhancing and/or change space expression and/or temporal expression.Promotor also can comprise far-end enhanser or repressor element, and it can be positioned at apart from transcription initiation site and reach several thousand base pairs.Promotor can stem from the source that comprises virus, bacterium, fungi, plant, insect and animal.Promotor can be according to the cell, tissue or the organ that wherein take place to express or according to the etap of taking place to express; Or in response to external stimulus for example physiological stress, pathogenic agent, metals ion or inductor come the composition of regulatory gene component, or differential expression.The representative example of promotor comprises phage t7 promotor, phage T3 promotor, SP6 promotor, lac operon-promotor, tac promotor, SV40 late promoter, SV40 early promoter, RSV-LTR promotor, CMV IE promotor, SV40 early promoter or SV40 late promoter and CMV IE promotor.

R. stringent hybridization condition

The condition that " stringent hybridization condition " that uses like this paper instigates first nucleotide sequence (for example, probe) and second nucleotide sequence (for example, target) in the complex mixture of for example nucleic acid, to be hybridized.Stringent condition be that sequence relies on and in different environment with difference.Stringent condition can be chosen to be under the ionic strength pH of regulation for specific sequence and be lower than pyrolysis chain temperature (T _m) about 5-10 ℃.T _mCan be 50% hybridize to target sequence (because the excessive existence of target sequence, at T with target complementary probe with equilibrium state _mThe place, 50% probe is occupied during balance) time temperature (the regulation ionic strength, pH and nucleic acid concentration under).Stringent condition can be when pH7.0 to 8.3 salt concn less than about 1.0M sodium ion; For example about 0.01-1.0M sodium ion (or other salt) concentration and temperature are for short probe (for example; About 10-50 Nucleotide) is at least about 30 ℃ and be about 60 ℃ condition for long probe (for example, greater than about 50 Nucleotide).Also can through add destabilizing agent for example methane amide reach stringent condition.For selectivity or specific hybrid, positive signal can be at least 2 to 10 times of background hybridization.Exemplary stringent hybridization condition comprises following: 50% methane amide, 5 * SSC and 1%SDS, under 42 ℃, hatch, or 5 * SSC, 1%SDS, under 65 ℃, hatch, in 0.2 * SSC and 0.1%SDS, washing under 65 ℃.

S. complementary basically

" complementary basically " of using like this paper refer to 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250 more a plurality of Nucleotide or 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,55,60,65,70,75,80,85 or 90 or more a plurality of amino acid whose zone in; The complementary strand of first sequence and second sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, is meant that perhaps two kinds of sequences hybridize under stringent hybridization condition.

T. same basically

" same basically " of using like this paper refer to 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250 more a plurality of Nucleotide or 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,55,60,65,70,75,80,85 or 90 or more a plurality of amino acid whose zone in; First and second sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity; Perhaps for nucleic acid, if first sequence is complementary to the complementary strand of second sequence basically.

U. variant

Be meant a part or the fragment of the nucleotide sequence that (i) quotes for nucleic acid like " variant " of this paper use; The complementary strand of the nucleotide sequence of (ii) quoting or its part; (iii) substantially the same in nucleic acid or its complementary strand of the nucleic acid of quoting; Or (iv) under stringent condition with the nucleic acid of the nucleic acid hybridization quoted, its complementary strand, or with its same basically sequence.

" variant " is meant through insertion, disappearance or preservative replacement amino acid for peptide or polypeptide and makes aminoacid sequence different, but keep at least a bioactive peptide or polypeptide.Variant can also be meant the protein that has basically with the same aminoacid sequence of the protein of quoting, and the said protein of quoting has at least a bioactive aminoacid sequence of reservation.Amino acid whose preservative replacement, promptly using the different aminoacids of similar performance (for example, the degree of wetting ability, charging zone and distribution) to substitute amino acid is known as the subtle change that typically relates in this area.As understanding in this area, can come partly to identify these subtle change through the wetting ability index of considered amino acid.Kyte etc., J.Mol.Biol.157:105-132 (1982).Amino acid whose wetting ability index is based on the consideration of its hydrophobicity and electric charge.Oneself knows the instead similar amino acid of wetting ability index and retaining protein function still in this area.On the one hand, replace and to have ± amino acid of 2 wetting ability index.Amino acid whose wetting ability also can be used for disclosing the displacement that will cause protein reservation biological function.Consider the hydrophilic amino acid in the peptide background; Allow to calculate the local average wetting ability (local average hydrophilicity) of maximum peptide; The USP 4 of all incorporating this paper into way of reference; 554,101 have reported useful measure so that antigenicity is well related with immunogenicity.As understanding in this area, the amino acid whose displacement with similar hydrophilicity value can cause peptide retains biological activity, for example immunogenicity.The amino acid of available hydrophilicity value each other in ± 2 is replaced.Amino acid whose wetting ability and hydrophilicity value all receive the influence of the special side chain of amino acid.Consistent with said observation, recognize that the amino-acid substitution compatible with biological function depends on amino acid, particularly the relative similarity of these amino acid whose side chains discloses as hydrophobicity, wetting ability, electric charge, size and other character.

V. carrier

The nucleotide sequence that refers to comprise replication orgin like " carrier " of this paper use.Said carrier can be carrier, phage, bacterium, artificial chromosome or yeast artificial chromosome.Carrier can be DNA or RNA carrier.Carrier can be the outer carrier of self-replacation karyomit(e), and is preferably the DNA plasmid.

2.RANTES

RANTES is coded by SEQ ID NO:1.Said sequence can further comprise one or more other aminoacid sequence elements.For example the RANTES by nucleic acid sequence encoding can further comprise IgE or the leading aminoacid sequence of IgG at its N-terminal.The leading aminoacid sequence of IgE can be SEQ ID NO:3.Likewise, the encoding sequence of coding IgE or IgG leader sequence can be connected to the immunogenic encoding sequence of coding.

Optimize SEQ ID NO:1 so that high level expression.Nucleotide sequence can comprise the Kozak sequence at 5 ' non-translational region.Nucleotide sequence can further comprise the nucleotide sequence of the leader sequence of encoding.The encoding sequence of N-terminal leader sequence is a 5 ' end of RANTES encoding sequence.The N-terminal leader can help secretion.The N-terminal leader can be IgE leader or IgG leader.SEQ ID NO:3 and SEQ ID NO:1 disclosed the same be the RANTES encoding sequence, further comprise the IgE encoding sequence; Leader sequence is connected to the proteinic N-terminal of RANTES.Nucleotide sequence can comprise the Kozak sequence at 5 ' non-translational region.SEQ ID NO:5 further comprises the Kozak sequence with the leading RANTES encoding sequence of the disclosed the same IgE of being of SEQ IDNO:3 and at 5 ' non-translational region.

3. genetic constructs and plasmid

This paper provides the genetic constructs of the nucleotide sequence that can comprise coding RANTES or its function fragment.The functional extrachromosomal molecule that genetic constructs can be used as the nucleic acid that comprises coding RANTES or its function fragment is present in the cell.The genetic constructs that comprises the nucleic acid of coding RANTES or its function fragment can be the minichromosome that contains kinetochore, telomere (telomer) or plasmid or clay.

Genetic constructs can also be a recombinant viral vector, comprises the genomic part of recombinant adenovirus, virus related to rocombinant adenovirus and recombinant vaccinia.Genetic constructs can be the part of genetic material in interior deactivation bacterium that survives of cell or the recombinant bacteria carrier.

Genetic constructs can comprise the controlling element of the genetic expression that is used for RANTES or its function fragment.Controlling element can be promotor, enhanser, initiator codon, terminator codon or polyadenylation signal.

Compsn can comprise first nucleotide sequence of coding RANTES or its function fragment, and can further comprise the one or more immunogenic nucleotide sequences of one or more other codings.First can be present on the identical nucleic acid molecule or different nucleic acid molecules with other nucleotide sequence.First can be controlled by at acting controlling element in people's cell with other nucleotide sequence.

It can be the genetic constructs of carrier that nucleotide sequence constitutes.Carrier can be to cause that effectively immunoreactive amount is at cell inner expression RANTES or its function fragment of individuality in the individuality.Carrier can be recombinated.Carrier can comprise the heterogeneous nucleic acid of coding RANTES or its function fragment.Carrier can be a plasmid.Said carrier can be used for using the nucleic acid transfection cell of coding RANTES or its function fragment, under the condition of RANTES or its function fragment generation expression, cultivates and the maintenance transformed host cells.

Carrier can comprise the heterogeneous nucleic acid of coding RANTES or its function fragment and can further comprise can be in the initiator codon at RANTES or its function fragment encoding sequence upper reaches and can be in the terminator codon in RANTES or its function fragment encoding sequence downstream.The initial sum terminator codon can with RANTES or its function fragment encoding sequence in frame (frame).Carrier also can comprise the promotor that is operably connected to RANTES or its function fragment encoding sequence.The promotor that is operably connected to RANTES or its function fragment encoding sequence can be the promotor from simian virus 40 (SV40); Mouse mammary tumour virus (MMTV) promotor; Human Immunodeficiency Viruses (HIV) promotor is long terminal repetition (LTR) promotor of ox immunodeficiency virus (BIV) for example; The moloney virus promotor; Avian leukosis viruses (ALV) promotor; Cytomegalovirus (CMV) promotor is the CMV immediate early promoter for example; Epstein-Barr virus (EBV) promotor or rous sarcoma virus (RSV) promotor.Promotor can be also being from the Human genome promotor of human actin, human myoglobulin, human hemoglobin, people's muscle creatine or human metal thioalbumen for example.Promotor can also be a tissue-specific promoter, for example muscle or skin specificity promoter, natural or synthetic.The case description of this promotor is in U.S. Patent Application Publication US20040175727 number, and its content is all incorporated this paper into.

Carrier also can comprise polyadenylation signal.They can be in the downstream of RANTES or its function fragment encoding sequence.Polyadenylation signal can be SV40 polyadenylation signal, LTR polyadenylation signal, Trobest (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal or people's betaglobulin polyadenylation signal.The SV40 polyadenylation signal can be from pCEP4 carrier (Invitrogen, San Diego San Diego, polyadenylation signal CA).

Carrier also can comprise enhanser at the upper reaches of RANTES or its function fragment encoding sequence.Enhanser can be that the DNA expression is necessary.Enhanser can be that human actin, human myoglobulin, human hemoglobin, people's muscle creatine or virus enhancer are for example from one of CMV, HA, RSV or EBV.At USP the 5th, 593, No. 972, the 5th, 962, No. 428 with WO94/016737 in the polynucleotide increased functionality has been described, its content separately integral body is by reference incorporated into.

Carrier also can comprise the Mammals replication orgin so that in cell, make carrier remain in outside the karyomit(e) and produce the copy of a plurality of carriers.Carrier can be that (it can comprise Epstein-Barr virus replication orgin and NA EBNA-1 coding region for San Diego, pVAX1 CA), pCEP4 or pREP4, and it can produce high copy episomal replication and unconformability from Invitrogen.The main chain of carrier can be pAV0242.Carrier can be replication defective sexual gland virus type 5 (Ad5) carrier.

Carrier also can comprise the adjusting sequence that can be adapted to the genetic expression in people's cell of using carrier to it well.Encoding sequence is more effectively transcribed in RANTES or its function fragment encoding sequence permission host cell.Carrier can be to produce protein expression carrier or system through routine techniques and the raw material that is easy to obtain; Comprise Sambrook etc.; Molecular Cloning A Laboratory Manual; Second Ed., Cold Spring Harbor (1989), it is all incorporated into by reference.

Also can be designed to have the immunogenic encoding sequence of coding except that RANTES or its function fragment for RANTES or the disclosed genetic constructs of its function fragment this paper and element, this construct will provide with the nucleotide sequence of coding RANTES or its function fragment and induce the vaccine that resists immunogenic immunoreactive improvement thus.

4. pharmaceutical composition

This paper provides according to pharmaceutical composition of the present invention, and it comprises the DNA of about 1 nanogram to about 10mg.In some embodiments; Pharmaceutical composition according to the present invention comprises from following 1) between and 2) between: 1) at least 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 nanograms; Or at least 1,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400,405,410,415,420,425,430,435,440,445,450,455,460,465,470,475,480,485,490,495,500,605,610,615,620,625,630,635,640,645,650,655,660,665,670,675,680,685,690,695,700,705,710,715,720,725,730,735,740,745,750,755,760,765,770,775,780,785,790,795,800,805,810,815,820,825,830,835,840,845,850,855,860,865,870,875,880,885,890,895.900,905,910,915,920,925,930,935,940,945,950,955,960,965,970,975,980,985,990,995 or 1000 micrograms, or at least 1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 or 10 milligrams or more more than; 2) up to and comprise 15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100; Or up to and comprise 1,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400,405,410,415,420,425,430,435,440,445,450,455,460,465,470,475,480,485,490,495,500,605,610,615,620,625,630,635,640,645,650,655,660,665,670,675,680,685,690,695,700,705,710,715,720,725,730,735,740,745,750,755,760,765,770,775,780,785,790,795,800,805,810,815,820,825,830,835,840,845,850,855,860,865,870,875,880,885,890,895.900,905,910,915,920,925,930,935,940,945,950,955,960,965,970,975,980,985,990,995 or 1000, or up to and comprise 1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 or 10 milligrams.In some embodiments, pharmaceutical composition according to the present invention comprises about 5 nanograms to about 10 milligrams DNA.In some embodiments, pharmaceutical composition according to the present invention comprises about 25 nanograms to about 5 milligrams DNA.In some embodiments, pharmaceutical composition comprises about 50 nanograms to about 1 milligram DNA.In some embodiments, pharmaceutical composition comprises about 0.1 DNA to about 500 micrograms.In some embodiments, pharmaceutical composition comprises about 1 DNA to about 350 micrograms.In some embodiments, pharmaceutical composition comprises about 5 DNA to about 250 micrograms.In some embodiments, pharmaceutical composition comprises about 10 DNA to about 200 micrograms.In some embodiments, pharmaceutical composition comprises about 15 DNA to about 150 micrograms.In some embodiments, pharmaceutical composition comprises about 20 DNA to about 100 micrograms.In some embodiments, pharmaceutical composition comprises about 25 DNA to about 75 micrograms.In some embodiments, pharmaceutical composition comprises about 30 DNA to about 50 micrograms.In some embodiments, pharmaceutical composition comprises about 35 DNA to about 40 micrograms.In some embodiments, pharmaceutical composition comprises about 100 to about 200 micrograms of DNA.In some embodiments, pharmaceutical composition comprises the DNA of about 10 micrograms to about 100 micrograms.In some embodiments, pharmaceutical composition comprises the DNA of about 20 micrograms to about 80 micrograms.In some embodiments, pharmaceutical composition comprises the DNA of about 25 micrograms to about 60 micrograms.In some embodiments, pharmaceutical composition comprises the DNA of about 30 nanograms to about 50 micrograms.In some embodiments, pharmaceutical composition comprises the DNA of about 35 nanograms to about 45 micrograms.In some preferred embodiments, pharmaceutical composition comprises about 0.1 to about 500 milligrams DNA.In some preferred embodiments, pharmaceutical composition comprises about 1 DNA to about 350 micrograms.In some preferred embodiments, pharmaceutical composition comprises about 25 DNA to about 250 micrograms.In some preferred embodiments, pharmaceutical composition comprises about 100 DNA to about 200 micrograms.

Pharmaceutical composition according to the present invention is prepared according to method of application to be used.At pharmaceutical composition is in the situation of Injectable composition, and they are aseptic, pyrogen-frees and do not have particulate.Preferred use waits oozes preparation (isotonic formulation).Usually, the isotonicity additive can comprise sodium-chlor, glucose, mannitol, sorbyl alcohol and lactose.In some cases, preferred isotonic solution phosphate buffered saline (PBS) for example.Stablizer comprises gelatin and BSA.In some embodiments, in preparation, add vasoconstrictor.

This paper provide can mediator's para-immunity the immunotherapeutic agent of reaction.Immunotherapeutic agent can comprise above-mentioned genetic constructs, and said genetic constructs comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or its function fragment.

This paper provides one or more immunogenic immunoreactive vaccines that can in the mankind, create antagonism.Said vaccine can comprise above-mentioned genetic constructs, and said genetic constructs comprises and above-mentioned SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 or its function fragment that contains the genetic constructs combination of the immunogenic nucleotide sequence of encoding.Coding RANTES or its segmental genetic constructs can with comprise on the identical or different nucleic acid molecule of the immunogenic nucleotide sequence of coding.

Immunotherapeutic agent or vaccine can be the DNA compsns.Said DNA compsn can comprise multiple identical or different plasmid, and said plasmid contains one or more genetic constructs.The DNA compsn can comprise the nucleotide sequence of coding RANTES or its function fragment.When it was vaccine, it can further comprise the immunogenic nucleotide sequence of coding on identical or different plasmid.

The dna vaccination technology that can be used for producing as the DNA plasmid compsn of immunotherapeutic agent or vaccine is disclosed in U.S. Patent number 5,593,972,5,739,118,5; 817,637,5,830,876,5,962; 428,5,981,505,5,580,859,5; 703,055 and 5,676,594, it all incorporates this paper by reference into.Dna vaccination can further comprise inhibition, and it is incorporated into chromosomal element or reagent.

Also can produce the recombinant viral vector that comprises above-mentioned genetic constructs.Recombinant viral vector also can comprise SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or its function fragment, has or do not have the genetic constructs that comprises the immunogenic nucleotide sequence of encoding.Can incorporate SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or its function fragment into attenuated vaccine, for example attenuated live vaccine, inactivated vaccine and use recombinant vectors are sent the vaccine of foreign gene.Attenuated live vaccine, use recombinant vectors send foreign gene those, the case description of subunit vaccine and glucoprotein vaccine is in U.S. Patent number: 4,510,245,4,797,368,4,722,848,4,790,987,4,920,209,5; 017,487,5,077,044,5,110,587,5,112,749,5,174,993,5,223; 424,5,225,336,5,240,703,5,242,829,5,294,441,5,294; 548,5,310,668,5,387,744,5,389,368,5,424,065,5,451; 499,5,453,364,5,462,734,5,470,734,5,474,935,5,482; 713,5,591,439,5,643,579,5,650,309,5,698,202,5,955; 088,6,034,298,6,042,836,6,156,319 and 6,589,529, it incorporates this paper separately by reference into.

Vaccine can further comprise pharmaceutically acceptable vehicle.Pharmaceutically acceptable vehicle can be the functional molecular such as vehicle, adjuvant, carrier or thinner.Pharmaceutically acceptable vehicle can be a transfection promotor, and it can comprise tensio-active agent, for example immunostimulating complex (ISCOMS), Freund's incomplete adjuvant (Freund ' s incomplete adjuvant), LPS analogue; Comprise monophosphoryl lipid A, muramylpeptides, quinone analogue, vesica be Supraene and Supraene for example; Mucinase, lipid, liposome, calcium ion; Virus protein, polyanion, polycation or nanoparticle, or other known transfection promotor.

Transfection promotor is polyanion, and polycation comprises and gathers L L-glutamic acid (LGS) or lipid.Transfection promotor is for gathering L L-glutamic acid, and more preferably gathers L L-glutamic acid and be present in the vaccine with the concentration less than 6mg/ml.Transfection promotor also can comprise for example immunostimulating complex (ISCOMS) of tensio-active agent; Freund's incomplete adjuvant, the LPS analogue comprises monophosphoryl lipid A, muramylpeptides; Quinone analogue and vesica be Supraene and Supraene for example, and also can mucinase and genetic constructs together be used.In some embodiments, DNA plasmid compsn also can comprise for example lipid of transfection promotor, liposome; Comprise lecithin liposome or other liposome known in the art (referring to for example WO9324640), calcium ion, virus protein as DNA-liposome mixture; Polyanion; Polycation, or nanoparticle, or other known transfection promotor.Preferably, transfection promotor is polyanion, and polycation comprises and gathers L L-glutamic acid (LGS), or lipid.The concentration of transfection agents is less than 4mg/ml, less than 2mg/ml, less than 1mg/ml, less than 0.750mg/ml, less than 0.500mg/ml, less than 0.250mg/ml, less than 0.100mg/ml, less than 0.050mg/ml or less than 0.010mg/ml in the vaccine.

Pharmaceutically acceptable vehicle can be an adjuvant.Adjuvant can be other gene of in optional plasmid, expressing or conduct and the protein delivery of above-mentioned plasmid combination.The group of the following composition of the optional freedom of adjuvant: alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, Thr6 PDGF BB (PDGF), TNF α, TNF β, GM-CSF, Urogastron (EGF), skin T cell are captured chemokine (CTACK), epithelium thymus gland and are expressed chemokine (TECK), epithelium chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 that mucous membrane is relevant, comprise that having disappearance signal sequence and optional comprises the IL-15 from the IgE signal peptide.Adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Thr6 PDGF BB (PDGF), TNF α, TNF β, GM-CSF, Urogastron (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 or its combination.

Other gene that can be useful adjuvant comprises those that coding is following: MCP-1; MIP-la; MIP-1p; IL-8; L-selects albumen; P-selects albumen; E-selects albumen; CD34; GlyCAM-1; MadCAM-1; LFA-1; VLA-1; Mac-1; Pl50.95; PECAM; ICAM-1; ICAM-2; ICAM-3; CD2; LFA-3; M-CSF; G-CSF; IL-4; The mutant form of IL-18; CD40; CD40L; Angiogenesis factor; Fibroblast growth factor; IL-7; NGFF; Vascular endothelial growth factor; Fas; The TNF acceptor; Flt; Apo-1; P55; WSL-1; DR3; TRAMP; Apo-3; AIR; LARD; NGRF; DR4; DR5; KILLER; TRAIL-R2; TRICK2; DR6; CaspaseICE; Fos; C-jun; Sp-1; Ap-1; Ap-2; P38; P65; Rel; MyD88; IRAK; TRAF6; IkB; Inactivation NIK; SAP K; SAP-1; JNK; The interferon response gene; NFkB; Bax; TRAIL; TRAILrec; TRAILrecDRC5; TRAIL-R3; TRAIL-R4; RANK; RANK LIGAND; Ox40; Ox40LIGAND; NKG2D; MICA; MICB; NKG2A; NKG2B; NKG2C; NKG2E; NKG2F; TAP1; TAP2 and function fragment thereof.

The plasmid compsn can further comprise the gene vaccine promotor described in the U. S. application sequence number 021,579 of submission in 1994 4 years 1 day, and it is all incorporated into by reference.

5. delivering method

This paper provides the method for delivering drugs preparation, so that coding RANTES or its segmental genetic constructs are provided, this genetic constructs has or do not have the genetic constructs of the immunogenic sequence that comprises coding expectation immunoreation antagonism.The method that the delivering drugs preparation can be provided is to regulate individual immunity system or to induce therapeutic and/or the preventative immunoreation that resists specific immunogens.

Can be with USP the 4th, 945, No. 050 and the 5th, 036, the form delivering compositions described in No. 006, both all incorporate into by reference.

A. use by way of

Can use compsn through different approach, comprise oral, parenteral, hypogloeeis, through skin, rectum, through mucous membrane, part, through suck, trans-oral is used, in the pleura, in the intravenously, intra-arterial, abdomen, in subcutaneous, the intramuscular, nose, in the sheath and intraarticular or its combination.Can pass through conventional syringe, needleless injection device, " microparticle bombardment particle gun " or other physical method for example electroporation (" EP "), " hydrodynamic method ", or ultrasonic.

The carrier of compsn can be delivered to Mammals through some well-known technology, said technology comprise that DNA injection (being also referred to as the DNA inoculation), this DNA injection have or not in vivo electroporation, liposome-mediated, nanoparticle promotes, recombinant vectors for example recombinant adenovirus, virus related to rocombinant adenovirus and recombinant vaccinia.

B. electroporation

Use via the DNA of the electroporation of vaccine plasmid and can use electroporation device to realize; Said electroporation device is configurable to be to send the energy pulse that causes in cytolemma that effectively reversible hole forms to the mammalian tissues of expectation, and the preferred energy pulse is the constant current that is similar to by the scheduled current of user's input.Electroporation device can comprise electroporation assembly and electrod assembly (electrode assembly) or processing element (handle assembly).The electroporation assembly can comprise and incorporate into one or more in the various elements of electroporation device, and said element comprises: RF, current waveform generator, impedance check, waveform log, input element, status report element, communication port, memory module, power supply and power switch.Can use in vivo electroporation device; (VGXPharmaceuticals of system for example; Blue Bell; PA) or Elgen electroporation device (Genetronics, San Diego CA) realize that electroporation is to promote the transfection via plasmid.

The effect of an element of electroporation assembly electrifiable perforating apparatus, and other element is the individual component (or assembly) with the electroporation component communication.The effect that surpasses an element of electroporation assembly electrifiable perforating apparatus, it still can be communicated by letter with other element from the electroporation device of electroporation components apart.As the element of the electroporation device of the part of motor or mechanical means can be not limited to the element that can play a kind of equipment or with the individual component of other devices communicating.The electroporation assembly can transmit the energy pulse that produces constant current in desirable tissue, and comprises Feedback mechanism.Electrod assembly can comprise having a plurality of steric electrod-arrays, and wherein electrod assembly is delivered to desirable tissue from the received energy pulse of electroporation assembly and through electrode with it.In a plurality of electrodes one of at least is neutral (neutral) during the transmission of energy pulse, and in desirable tissue, measures impedance and impedance is communicated to the electroporation assembly.Feedback mechanism can receive measured impedance and adjustable by the energy pulse of electroporation component passes to keep constant current.

A plurality of electrodes can transmit energy pulse with dispersion pattern.A plurality of electrodes can transmit energy pulse with the dispersion pattern of controlling through the electrode under program (programmed sequence), and said program is input to the electroporation assembly by the user.Said program can comprise a plurality of pulses of transmitting successively; Each pulse in wherein a plurality of pulses is transmitted by two active electrodes (active electrode) with neutral electrode measuring impedance at least, and in wherein a plurality of pulse subsequently pulse by transmitting with the different electrode of at least two active electrodes with neutral electrode of measuring impedance.

Feedback mechanism can be through perhaps hardware or software carry out.Feedback mechanism can carry out through the analog closed-loop circuit.Per 50 μ s, 20 μ s, 10 μ s or 1 μ s feed back, but preferred in real time or instantaneous feedback (that is, basically by measure the obtainable technical measurement of time of response time).Neutral electrode can be measured the impedance in the expectation tissue and impedance is communicated to Feedback mechanism, and Feedback mechanism responds to impedance and regulates energy pulse so that constant current remains in the value of similar scheduled current.Feedback mechanism during energy pulse transmits serially with keep constant current instantaneously.

Can promote the instance of electroporation device that dna vaccination of the present invention is sent and electroporation method to be included in the USP the 7th of Draghia-Akli etc.; 245; Described in the U.S. Patent Publication 2005/0052630 that No. 963, Smith etc. are submitted to those, its content in view of the above by reference integral body incorporate into.In No. the 11/874072nd, can be used for promoting other electroporation device that dna vaccination is sent and electroporation method to comprise being provided in submitting on October 17th, 2007 common co-pending and the U.S. Patent application sequence owned together those; It requires the U.S. Provisional Application sequence number 60/852 of submission on October 17th, 2006 according to 35USC 119 (e); 60/978 of submission on October 10th, 149 and 2007; 982 rights and interests, its all the elements integral body are in view of the above incorporated into.

The USP of Draghia-Akli etc. has been described the standard electrode system for the 7th, 245, No. 963 and has been used for promoting biomolecules to import in the body or the purposes of the selected cells of tissues of plant.The standard electrode system can comprise a plurality of pin electrodes; Hypodermic needle; Provide from the plug of constant current pulses RF able to programme to the electroconductibility connection of a plurality of pin electrodes; And power supply.The operator can hold a plurality of pin electrodes of being fixed on the bearing structure and be inserted in the body securely or the selected tissue of plant.Biomolecules is delivered to selected tissue via hypodermic needle then.Activate constant current pulses RF able to programme and the constant current electricimpulse is applied to a plurality of pin electrodes.The constant current electricimpulse that applies promotes the biological cell that imports between a plurality of electrodes that divided.USP the 7th, 245, No. 963 full content in view of the above by reference integral body incorporate into.

U.S. Patent Publication 2005/0052630 description by submissions such as Smith can be used for promoting effectively biomolecules to import in the body or the electroporation device of the selected cells of tissues of plant.Said electroporation device comprises by the electrical equipment of software or its operation of firmware specifications (" EKD equipment ").EKD equipment produces a series of constant current pulses patterns able to programme based on the input of user's control and pulse parameter between the electrode of array, and allows storage and obtain the current waveform data.Electroporation device comprises that also removable electrode plate with pin electrode array, entry needle are with center injection passage and removable positioning disk.With the full content of U.S. Patent Publication 2005/0052630 in view of the above by reference integral body incorporate into.

USP the 7th, 245 not only can be suitable for deep penetration to organizing for example muscle with electrod-array and the method described in the U.S. Patent Publication 2005/0052630 No. 963, and can be suitable for deep penetration and arrive other tissue or organ.Because the structure of electrod-array, entry needle (being used to send selected biomolecules) also is inserted into the target organ fully, and in the zone of being described in advance by electrode, injects perpendicular to said target spot.USP the 7th, 245 disclose the electrode described in 2005/005263 with USP and is preferably that 20mm grows and 21gauge for No. 963.

In addition, comprised electroporation device of incorporating into and uses thereof in some embodiments, electroporation device has those described in the following patent: the USP 5,273 that on December 28th, 1993 announced; 525,6 of the USP of announcing on August 29th, 2,000 6,110,161, announcement on July 17 calendar year 2001; 6,958 of announcement on October 25th, 261,281 and 2005; The USP 6,939,862 that on September 6th, 060 and 2005 announced.In addition, the patent that contains theme is provided in the USP 6,697 announced on February 24th, 2004; 669, it relates to any DNA delivery used in the plurality of devices and the USP of announcing on February 5th, 2,008 7; 328,064 have described the method for the injection DNA that this paper comprises.The mode that above-mentioned patent is quoted with its integral body is incorporated into.

C. the preparation method of plasmid

This paper provides the preparation method of the DNA plasmid that comprises SEQ ID NO:1 described herein.DNA plasmid (in the end getting into the Mammals expression plasmid after the subclone step) can be used for using the currently known methods inoculating cell culture in large fermentation tank in this area.

Though the DNA plasmid that supplies EP equipment of the present invention to use can use the known device and the combination of technology to prepare or make; But the plasmid manufacturing technology of the optimization described in the co-pending U.S. Provisional Application United States serial 60/939, the 792 preferred usage license, that on March 23rd, 2007 submitted to is made them.In certain embodiments, the DNA plasmid that in these researchs, uses can be prepared with the concentration more than or equal to 10mg/mL.Manufacturing technology also comprises or incorporates into various device and rules; These equipment and rules are conventional known to the one of ordinary skilled in the art; Except United States serial 60/939792 described those; Comprise described in No. the 7th, 238,522, the permission patent USP announced on July 3rd, 2007 those.Incorporate above-mentioned application of quoting and patent, United States serial 60/939,792 into this paper respectively with its integral body in view of the above the 7th, 238, No. 522 with USP.

6. immunogen

The present invention can be used for immune body with enantiopathy substance for example virus, prokaryotic organism and for example unicellular pathogenic organisms of pathogenic eukaryote and many cells parasite.The present invention is used in particular for immune body with antagonism cells infected and acapsular those pathogenic agent for example gonorrhoea bacterium, listeria and Shigella of virus and prokaryotic organism for example.In addition, the present invention also is used for immune body with antagonism protozoon pathogenic agent, and said protozoon pathogenic agent comprises that they are the life cycle phase of intra-cellular pathogens.Table 1 provides and can prepare according to some Viraceae of vaccine of the present invention and the tabulation of genus.The DNA construct that comprises the dna sequence dna of encoded peptide is used for vaccine, and said peptide comprises the epi-position that is equal to or is substantially similar to the epi-position that shows on those listed in pathogen antigen is for example shown antigens at least.In addition, the present invention also is used for immune body to resist other pathogenic agent, comprises protokaryon and eucaryon protozoon pathogenic agent and many cells parasite example is as shown in table 2 those.

Table 1-virus

Picornaviridae

Belong to:

Rhinovirus: (medical science) is the cause of disease of common cold case-50%.

Enterovirus (Etheroviruses): (medical science) comprises for example hepatitis A virus of poliomyelitis virus, Coxsackie virus, Echo virus and human enterovirus.

Blue tongue virus (Apthoviruses): they are foot and mouth disease viruses for (animal doctors).

Target antigen: VP1, VP2, VP3, VP4, VPG

Calicivirus (Calcivirus) section

Belong to:

C (Norwalk) group of virus: (medical science) these viruses are the important pathogenic agent of epidemic gastroenteritis.

Togavirus (Togavirus) section

Belong to:

Alphavirus (Alphaviruses): (medical science and animal doctor) instance comprises sindbis virus (Sindbis virus), ross river virus (RossRiver virus) and Venezuela's thing equine encephalitis virus (Venezuelan Eastern Western Equine encephalitis).

Reovirus (Revirus): (medical science) rubella virus.

Flavivirus (Flariviridae) section

Instance comprises: (medical science) singapore hemorrhagic fever, yellow jack, Japanese encephalitis, st. louis encephalitis and tick-brone encephalitis virus.West Nile virus (Genbank NC001563, AF533540, AF404757, AF404756, AF404755, AF404754, AF404753, AF481864, M12294, AF317203, AF196835, AF260969, AF260968, AF260967, AF206518 and AF202541)

Representative target antigen: E NS5C

Hepatitis C virus: (medical science) these viruses are not included into same section, but think one of togavirus or flavivirus.Section is the most similar with togavirus.

Coronaviridae: (medical science and animal doctor)

IBV (fowl)

Transmissible gastro-enteritis virus (pig)

Pig hemagglutination encephalomyelitis virus (pig)

Feline infectious peritonitis virus (cat)

Cat enteric coronavirus virus (cat)

Dog coronavirus (dog)

The coronavirus that SARS is relevant

The human respiratory coronavirus causes about 40% common cold case.EX.224E, OC43Note coronavirus can cause non-A, B or C hepatitis.

Target antigen: El-is also referred to as M or stroma protein E2-and is also referred to as S or spike protein-be also referred to as BE or hemagglutinin-elterose gp (all not existing in the coronavirus) N-nucleocapsid

Rhabdoviridae

Belong to:

Bubble venereal disease poison, rabies virus: (medical science and animal doctor) rabies

Target antigen: G albumen, N albumen

Filoviridae: (medical science)

Hemorrhagic fever virus is horse Burger and Ebola virus for example

Paramyxoviridae:

Belong to:

Paramyxovirus: (medical science and animal doctor) mumps virus, NDV (the important pathogenic agent in the chicken)

Measles virus: (medical science and animal doctor) measles, distemper

Pneumonitis virus: (medical science and animal doctor) respiratory syncytial virus

Orthomyxoviridae family's (medical science) influenza virus

Bunyavirus (Bunyavirus) section

Belong to:

Bunyavirus: (medical science) galifornia encephalitis, La Crosse virus (La Crosse)

Sand fly virus (Phlebovirus): (medical science) enzootic hepatitis (Rift Valley Fever)

Hantaan virus: pounce on heat and draw (Puremala) to be hemahagin fever virus (hemahagin fever virus)

How grace virus (Nairvirus) (animal doctor) Nairobi sheep disease (Nairobi sheep disease)

Also have many unnamed bunyaviruss (bungaviruses)

Sand grains Viraceae (medical science) LCM, Lassa fever virus (Lassa fever virus)

Reoviridae

Belong to:

Reovirus: potential human pathogen

Rotavirus: children acute gastro-enteritis

Orbivirus: (medical science and animal doctor) colorado tick fever (Colorado Tick fever),

Lebombo virus (Lebombo) (people) horse encephalosis, bluetongue

Retroviridae

Subfamily:

Oncorivirinal: (animal doctor) (medical science) cat leukemia virus, HTLVI and HTLVII

Slow virus (Lentivirinal): (medical science and animal doctor) HIV, cat immunodeficiency virus, horse infect, anemia virus

Spumavirinal papovavirus section

Subfamily:

Polyomavirus: (medical science) BKU and JCU virus

Subfamily:

Papilloma virus: (medical science) many Virus Types are relevant with the deterioration development of cancer or papilloma.

Adenovirus (medical science) EX AD7, ARD., O.B.-cause respiratory tract disease-some adenovirus for example 275 to cause enteritis Parvoviridae (animal doctor)

Feline panleucopenia virus: cause feline enteritis

Cat infectivity agranulocytosis virus (Feline panleucopeniavirus)

Canine parvovirus

Pig parvoviral

Herpetoviridae

Subfamily:

The α herpetoviridae

Belong to:

Hsv (medical science)

HSVI(Genbank?X14112、NC001806)，

HSVII(NC001798)

Varicella zoster (Varicella zoster): (medical science animal doctor)

Pseudorabies

Varicella zoster

Subfamily:

The β herpetoviridae

Belong to:

Cytomegalovirus (medical science)

HCMV

MCMV

Subfamily

Gamma herpes viruses section

Belong to:

Lymphocryptovirus (medical science)

EBV-(the Bai Jili lymphoma (Burkitt ' slymphoma))

Variola virus section

Subfamily:

Chordopoxvirinae (Chordopoxviridae) (medical science-animal doctor) belongs to:

Smallpox (variola)

Acne rash (cowpox)

Parapoxvirus (Parapoxivirus)-animal doctor

Fowlpox virus (Auipoxvirus)-animal doctor

Goat capripoxvirus (Capripoxvirus)

Hare poxvirus (Leporipoxvirus)

Pig pox virus (Suipoxvirus)

Subfamily:

Entemopoxviridue

Hepadnaviridae

Hepatitis B virus

Non-classified hepatitis D virus

Table 2

Bacterial pathogen

Pathogenic GPC comprises: streptococcus pneumoniae, staphylococcus and suis.

Pathogenic gram-negative coccus comprises: meningococcus and gonococcus.

Pathogenic enteron aisle is removed from office blue bacillus and is comprised: enterobacteriaceae; Pseudomonas, positive acinetobacter calcoaceticus (acinetobacteria) and dust are agree bacterium (eikenella), pseudoglanders; Salmonellas; Shigellosis; Influenzae; Venereal ulcer; Brucellosis (brucellosis); Tularemia; Yersinia (Pasteurella); Streptobacillus moniliformis and spirobacteria; Listeria monocytogenes; Erysipelothrix ruhsiopathiae; Diphtheria, cholera, anthrax; Sick (donovanosis) (venereal granuloma) of Du Nuofan; And carrion's disease (bartonellosis).

Pathogenic anerobes comprises: tetanus; Clostridium botulinum (botulism); Other clostridium; White plaque; And other mycobacterium.

Pathogenic spirochetosis comprises: syphilis;-treponematosis; Yaws (yaws), pinta (pinta) and endemic syphilis (endemic syphilis); And leptospirosis.

The infection of other that is caused by senior pathogenic agent bacterium and pathomycete comprises: actinomycosis; Nocardiosis; Torulosis; Torula yeast is sick; Darling's disease and ball mycosis; Candidiasis, aspergillosis and mucormycosis; Sporotrichosis; Paracoccidioidomycosis (paracoccidiodomycosis), Podbielniak mycosis (petriellidiosis), torulopsis (torulopsosis), mycetoma (mycetoma) and chromomycosis (chromomycosis); And tinea.

Rickettsial infection comprises Rickettsiae microorganism belonging to genus (rickettsial) and rickettsiosis (rickettsioses).

The instance of mycoplasma and choamydiae infection comprises: mycoplasma pneumonia (mycoplasma pneurnoniae); Lymphogranuloma venereum; Psittacosis; With choamydiae infection perinatal period.

Pathogenic eukaryote

Pathogenic protozoon and worm and infection thus comprise: loeschiasis; Malaria; Leishmaniasis; Trypanosomiasis; Toxoplasmosis; Pneumocystis pneumoniae (pneumocystis carinii); Babesiosis; Chronic dysentery; Trichonematosis; Filaricide; Schistosomicide; Nematode; Leech (trematodes) or fluke (flukes); Tapeworm (cestode) (band worm (tapeworm)) infects.

Another aspect of the present invention provides the method for giving the protective immunological reaction that resists the excessive proliferated cell that is characterized as excess proliferative disease and provides treatment to suffer from the method for the individuality of excess proliferative disease.The instance of excess proliferative disease comprises the cancer and the psoriasis of form of ownership.

The genetic constructs of having found to contain the nucleotide sequence of coding immunogenicity " excessive proliferated cell " related protein imports in the individual cells that individual cells causes inoculating and produces those protein.For immunity with the antagonism excess proliferative disease, use the genetic constructs that comprises the coding proteinic nucleotide sequence relevant with excess proliferative disease to individuality.

In order to make the relevant protein of excess proliferative become effective immunogenicity target, it is necessary for compares in the one property generation of hyper-proliferative sexual cell special secondary school with normal cell or is in higher levels of protein.Target antigen comprises this proteinoid, its fragment and peptide; It is included in the epi-position of finding on this protein at least.In some cases, the protein that excess proliferative is relevant is the product of the gene mutation body of coded protein.The mutator gene coding causes undiscovered slightly different aminoacid sequence on the normal protein matter, almost being equal to the protein of normal protein matter except having.This target protein comprises by oncogene those of myb, myc, fyn and transposition gene bcr/abl, ras, src, P53, neu, trk and EGRF encoded protein matter for example.Except the oncogene product as the target antigen; The target protein that is used for anticancer therapy and preventative scheme comprises the variable region through the TXi Baoshouti of the variable region of the antibody of B cell lymphoma preparation and t cell lymphoma, and it also is used as the target antigen of autoimmune disease in some embodiments.Can use the relevant protein of other tumour as the protein that target protein for example exists with high level in tumour cell, comprise protein and FABP or PSA by monoclonal antibody 17-IA identification.

Though the present invention can be used for immune body to resist in some cancer forms one or more, the present invention is used for the individuality that preventative immunity is tended to develop particular cancers or had cancer and therefore be easy to recur especially.Development in genetics and technic and the epidemiology allows to measure the probability and the risk assessment of cancer development in the individuality.Utilize genetic screening and/or family's health history, can predict the probability any in some types of cancer that develops into that particular individual has.

Similarly, have being easy to recurrence especially and sending out again of the cancer that developed with having treated to remove those individualities cancer or that be in the alleviation with other form.As the part of regimen, can immunity should individuality be diagnosed as cancer so that antagonism is sent out again with them with antagonism.Therefore, in case known individuality has had one type cancer and has been in the risk of recurrence, then can carry out immunity so that its immunity system is prepared the cancer that antagonism occurs any future to it.

The present invention provides treatment to suffer from the method for the individuality of excess proliferative disease.In these class methods, the importing of genetic constructs is as immunotherapeutic agent, and guidance and promotion individual immunity system produce the hyper-proliferative sexual cell of target protein with antagonism.

When treatment or preventing cancer, the embodiment that does not have cell is useful especially.

The present invention provides through giving the sessile protective immunological reaction suffers from the method for the autoimmune disease and the individuality of disorder with the antagonism target relevant with autoimmunity (cell that comprises cell receptor and generation self-conductance antibody (self-directed antibodies) comes) treatment.

The cell-mediated autoimmune disease of T comprises rheumatoid arthritis (RA); Multiple sclerosis (MS); Sjogren syndrome (Sjogren's syndrome); Sarcoidosis; Insulin-dependent diabetes mellitus (IDDM); The autoimmunization thyroiditis; Reactive arthritis; Ankylosing spondylitis; Scleroderma; Polymyositis; Dermatomyositis; Psoriasis; Vasculitis; Wei Genashi granulomatosis (Wegener's granulomatosis); Crohn's disease (Crohn's disease) and ulcerative colitis.In these diseases each is characterised in that the TXi Baoshouti inflammatory cascade relevant with autoimmune disease with initiation that is attached to endogenous antigen.Inoculation to T cell variable region will cause that immunoreation comprises that CTL is to eliminate those T cells.

In RA, the variable region of the specific t-cell receptor of some involved in diseases (TCR) characterized.These TCR comprise V. β .-3, V. β .-14,20V. β .-17 and Va-17.Therefore, will cause the immunoreation of target being participated in the T cell of RA with these proteinic at least a DNA construct inoculations of coding.Referring to, Howell, M.D etc., 1991Proc.Nat.Acad.Sci.USA 88:10921-10925; Piliard, X etc., 1991Science 253:325-329; Williams, W.V etc., 1992J Clin.Invest.90:326-333; It incorporates this paper separately by reference into.In MS, the specificity variable region of the TCR of some involved in diseases is characterized.These TCR comprise VfP and Va-10.Therefore, will cause the immunoreation of target being participated in the T cell of MS with these proteinic at least a DNA construct inoculations of coding.Referring to: Wucherpfennig, K.W etc., 1990Science 248:1016-1019; Oksenberg, J.R etc., 1990Nature 345:344-346; It incorporates this paper separately by reference into.

In scleroderma, the specificity variable region of the TCR of some involved in diseases is characterized.These TCR comprise V. β .-6, V. β .-8, V. β .-14 and Va-16, Va-3C, Va-7, Va-14, Va-15, Va-16, Va-28 and Va-12.Therefore, will cause the immunoreation of target being participated in the T cell of scleroderma with these proteinic at least a DNA construct inoculations of coding.

In order to treat the patient who suffers from the cell-mediated autoimmune disease of T, particularly the TCR variable region does not also have those of sign, can carry out synovial biopsy.Can adopt existing T cell sample and the variable region of using standard technique to identify those TCR.Use this information can prepare gene vaccine.

The cell-mediated autoimmune disease of B comprises lupus (SLE), grave disease (Grave's disease), myasthenia gravis, autoimmune hemolytic anemia, autoimmunization aleukia, asthma, cryoglobulinemia, primary biliary cirrhosis and pernicious anemia.Each of these diseases is characterised in that the antibody inflammatory cascade relevant with autoimmune disease with initiation that is attached to endogenous antigen.Inoculation to the variable region of antibody will cause that immunoreation comprises that CTL is to eliminate those B cells that produce antibody.

In order to treat the patient who suffers from the cell-mediated autoimmune disease of B, must identify the variable region of participating in the active antibody of autoimmunization.Examination of living tissue can be carried out and existing antibody sample can be adopted in the inflammation site.The variable region of those antibody can use standard technique to identify.Use this information can prepare gene vaccine.

With regard to SLE, think that a kind of antigen is DNA.Therefore, in treating the patient of immunity, can screen their serum, and can prepare the vaccine of the DNA construct of the variable region that comprises this ADA of finding in the coding serum to ADA with antagonism SLE.

Common structure characteristic in the variable region of TCR and antibody is well-known.The dna sequence dna of coding specific T CR or antibody usually can according to well-known method for example Kabat equal Sequence of Proteins of Immunological Interest U.S.Department of Health and Human Services in 1987; Described those methods of Bethesda Md find, and the document is incorporated this paper by reference into.In addition, be found in Chaudhary from the general method in the function-variable district of antibody for the clone, V..K etc., 1990Proc.Natl.Acad Sci.USA 87:1066, it incorporates this paper by reference into.

But except the expression-form that uses the immune modulator encoding sequence to improve the gene vaccine, the invention still further relates to and use recombinant vectors with the attenuated live vaccine of the improvement of the foreign gene of sending coding for antigens and the vaccine of improvement.Attenuated live vaccine with use recombinant vectors with those the case description of sending exogenous antigen in U.S. Patent number: 4,722,848,5,017,487,5,077,044,5,110,587,5; 112,749,5,174,993,5,223,424,5,225,336,5,240; 703,5,242,829,5,294,441,5,294,548,5,310,668,5; 387,744,5,389,368,5,424,065,5,451,499,5,453; 364, in 5,462,734,5,470,734 and 5,482,713, it incorporates this paper separately by reference into.Gene construct is provided, and it comprises the nucleotide sequence of coding IL-28 or its function fragment, and wherein nucleotide sequence is operably connected to the adjusting sequence that can in vaccine, do in order to the influence expression.Gene construct is mixed attenuated live vaccine and recombiant vaccine to produce the vaccine according to improvement of the present invention.

The present invention provides the improvement method of immune body, and it comprises the step that arrives individual cells as a part of delivery of gene construct of the vaccine composition that comprises dna vaccination, attenuated live vaccine and recombiant vaccine.Gene construct comprises coding IL-28 or function fragment and is operably connected to the nucleotide sequence that can in vaccine, do in order to the adjusting sequence of influence expression.The vaccine that improves causes the enhanced cell immunoreation.

Embodiment

Embodiment 1

In following examples, further specify the present invention.Show the preferred embodiments of the invention though should understand present embodiment, only the mode with explanation provides.From above argumentation and present embodiment, those skilled in the art can confirm essential feature of the present invention, and under the situation that does not deviate from its spirit and scope, can make variations and modifications so that it is adapted to various uses and condition to the present invention.Therefore, from above description, except that will being conspicuous with described of the present invention various modifications those as far as those skilled in the art shown in this paper.This type of modification also is intended to fall in the scope of accompanying claims.

Foreword

Optimization electroporation (EP) the inductive cell immune response of the common SIVmac plasmid construction body of evaluation expression SIVgag, env and pol; And itself and optimization electroporation (EP) inductive cell immune response with the common SIVmac plasmid construction of the EP bonded body of the rhesus monkey IL-12 of expression plasmid compared, or RANTES can change the size and the quality of cell immune response.The deep improvement of t cell immune response is observed multi-functional flow analysis as measuring through ELISpot, vitro proliferation and epi-position width.These inoculation methods have also been carried out to impact assessment coupling not, the virus load of SIVmac251 internal rectum in exciting.Find that the virus that independent DNA inoculation influences in this research excites and prevent the cd4 t cell loss.The co-delivered of plasmid IL-12 and RANTES causes the enhancing of virus replication control.Therefore, handle simultaneously plasmid vaccine box (cassette) as if send with the generation that designs for the high frequency cell immune response relevant with the HIV model system be important.

Material and method

Animal.Standard according to U.S. laboratory animal management approval association (American Association for Accreditation of Laboratory Animal Care); With coming from Chinese rhesus monkey (Macaca mulatta) stable breeding in BIOQUAL; Inc. (Rockville, MD).Animal is isolated adapt at least 30 days.

Immunity.With each the rhesus monkey (DNA) among 1.5mg SIVgag, SIVenv and the SIVpol six one group of 0,8,12 and 24 week immunity.To be delivered to the single site in the musculus quadriceps through EP in vivo at the DNA of each immune time point.With SIVgag, SIVenv, SIVpol and the rhesus monkey IL-12 (DNA+12) of 1.5mg or among the RANTES (DNA+RANTES) each other two groups of one group of six rhesus monkey of 0,8 and 12 week immunity.In DNA+12 and DNA+RANTES group, from the 4th immunity (24 week), get rid of the plasmid adjuvant.With sterile water for injection as six rhesus monkeies of negative control (Naive) immunity.Use constant current equipment (VGX Pharmaceuticals; The Woodlands TX) carries out whole EP steps.The EP condition is 0.5Amp, 3 subpulses, 52 milliseconds of pws, 1 second recurrent interval.

Blood collection.Per two weeks draw blood to animal during the research.The 10ml blood collection is managed in EDTA.Centrifugal and be resuspended in perfect medium (RPMI 1640, have the 2mM L-glutaminate, are supplemented with 10% heat inactivation foetal calf serum, 100IU/ml penicillium mould, 100 μ g/ml Streptomycin sulphates and 55 μ M beta-mercaptoethanols through standard ficoll-Hai Parker (hypaque).) in separate PMNC (PBMC).(Cambrex Bio Science, East Rutherford NJ) come cracking RBC with the ACK lysis buffer.

Plasmid and plasmid product.The plasmid that uses in this research is expressed the total antigen of modifying for SIVgag (pSIVgag), SIVpol (pSIVpol) or SIVenv (pSIVenv).In order to improve expression, comprise effective IgE leader sequence.In addition, shorten SIVenv V1 and V2 zone and block the kytoplasm tail through the glycosylation position of removing the N connection with prevention adventitia recycling (envelope recycling).For SIV pol, introduce 3 sudden changes so that proteolytic enzyme, reversed transcriptive enzyme and RNAseH inactivation.The SIV dna immunization of gained optimization was codon and RNA optimize, synthetic originally, and was cloned into the pVAX1 expression vector to produce the expression construct that SIVgag (pSIVgag), SIVenv (pSIVenv) and SIVpol (pSIVpol) optimize.(the Woodlands TX), and prepares to be used for research described herein for VGXI, Inc. with these plasmid construction bodies of 10L macro preparation then.The DNA of purifying is formulated in the citrate buffer (pH6.7) of the 0.15M that in water, has 0.25% fourth croak cacaine.

Peptide.Pass through NIH; NIAID, the AIDS research and the reference reagent plan (AIDS Research and Reference Reagent Program) of AIDS department obtain reagent: SIVmac239gag Peptides (#6204), SIVmac 239env Peptides (#6883) and SIVmac239pol Peptides (#6443).

Fluoresceincarboxylic acid succinimido ester (CFSE) conjugation and the flow cytometry of PBMC.On the 4th isolating fresh PBMC of two week of immunity back, carrying out CFSE as previously mentioned.

Enzyme linked immunological engram analysis (ELISpot).Use monkey IFN ELISpot kit (Mabtech, Cincinnati, OH), like the ELISpot that carries out noted earlier.Input cell count for preceding twice immunity is 2 * 10 ⁵Individual cell.For third and fourth immune time point, because excessive spot number makes the input cell count be reduced to 1 * 10 ⁵Individual cell.Adjustment from the mean number of spot in the in triplicate hole to reflect per 10 ⁶The spot number of individual PBMC.The value of the spot that forms from each SIV peptide storehouse that basis is used for stimulating deducts according to calculating the SIV specific reaction behind the spot of single culture base formation.To be>=50SFU/10 ⁶PBMC and be regarded as the positive greater than the reaction of 2 * single culture radical reaction, and increase net value then.

The mapping of SIVpol epi-position matrix.As noted earlier, use matrix format (Matrix format) that the SIVpol peptide library is divided into 33 storehouses, and be used to stimulate the PBMC of the 4th the immunity isolating freezing preservation in back.Use IFN ELISpot to analyze the evaluation response width.Estimate the maximum quantity of epi-position based on the sum of the peptide of discerning.Through with the quantity of positive peptide stream (runs) (a series of two or more continuous positive peptides) divided by 3 and be rounded to the minimum that immediate integer calculates epi-position.

Intracellular cytokine dyeing.After like aforementioned the 4th 2 weeks of immunity back and immunity at last, carried out multi-functional T cell analysis on the isolating fresh PBMC in 8 months.Report data after the background correction.Positive reaction is defined as greater than 0.05%.

Leave standstill with the PBMC quick-thawing of freezing preservation and in 37 ℃ perfect medium, spending the night after the stimulation.On the antigen-specific cd8 t cell, measure each function and repertoire reaction that CCR5 (BD Biosciences) expresses.IFN and TNF Alpha antibodies are placed on the Alexa Fluor 700 to regulate dyeing other in this group.Report data and be shown as the percentages show of total CD8 colony after the background correction.

SIVmac251 excites.Back 8 months SIVmac251 with 25 monkey infective doses (MID) excite animal through the internal rectum approach the 4th immunity.By Ronald Desrosiers preparation and by Advanced BioSciences Laboratories, (ABL, Inc.) (Kensington MD) provides virus stock solution used and by BIOQUAL, Inc. (Rockville, MD) titration to Inc.

To plasma viral load quantitatively based on the analysis of the amplification (NASBA) of nucleotide sequence.As noted earlier, analyze SIVgag by ABL Inc. through quantitative NASBA and measure plasma viral load.

MHCI genoid type somatotype uses the combination of aforementioned Sanger order-checking and tetra-sodium sequence measurement to carry out the MHC-I genotyping based on comprehensive sequence.In brief, (Roche Applied Sciences, Indianapolis IN) separates total cell RNA from PBMC to use MagNA Pure LC RNA separating kit.Use the SuperscriptIII first chain synthesis system (Invitrogen; Carlsbad CA) produces cDNA by RNA, and as using Phusion high-fidelity polysaccharase (New England BioLabs; Ipswich is MA) with the template of the pcr amplification of MHC-I special primer.(Qiagen, Valencia CA) come gel-purified PCR product to use the MinElute gel to reclaim test kit.For Sanger order-checking, use have the colibacillary Zero Blunt of TOP10 chemoreception attitude TOPO PCR clone test kit (Invitrogen, Carlsbad, CA) with these product cloning in bacterium.Every animal has 96-192 bacterial colony overnight growth in LB+50 μ g/ml kantlex that transforms; Use Perfectprep plasmid 96Vac Bind test kit (5PRIME, Gaithersburg, MD) isolated plasmid dna subsequently.Use sequence-specific PCR primer to utilize DYEnamic ET terminator cycle sequencing test kit (GE Healthcare; Piscataway; NJ) cover the Sanger sequencing reaction of alterable height peptide-binding domains by MHC-I exon 2 and 3 codings; And (Applied Biosystems, Foster City CA) analyze to use Applied Biosystems 3730xl Genetic Analyzer.Use the Roche/454-tetra-sodium order-checking of universal diagnostic 190bp pcr amplification in the MHC-I exon 2 grouping of 5 animals of having gone back genotyping.This amplicon by cDNA amplification, gel-purified, quantitatively, stdn and build the storehouse and at 454 order-checking center (454Life Sciences; Branford; CT) (Urbana moves on gene order-checking appearance FLX instrument IL) at the Urbana-Champaign high-flux sequence center of university or in the Illinois.(MA) (DNA Star, Madison WI) carry out sequential analysis with Lasergene 8 for CodonCode Corporation, Deham to utilize CodonCode aligner.Use BLASTN that the contig of assembling is compared with the allelic internal database of known rhesus monkey MHC-I.

Statistics.The comparison that produces for IFN γ ELISpot, T cell proliferation and cytokine, the unidirectional ANOVA that when suitable use SPSS17.0 statistical software, carries out two tail Mann-Whitney checks or check afterwards by means of Tukey or DunnettT3.Before statistical study, virus load is carried out number conversion with standardized data.< 0.05 P value is considered to significant.

The result

Research and design.24 rhesus monkeies that come from China are divided into four immune group (table 3).Group 1 (DNA) accepts the optimization plasmid of coding SIVgag, SIVenv and SIVpol.Group 2 (DNA+12) accept the plasmid of coding rhIL-12 and group 3 (DNA+RANTES) accept coding RANTES except that the SIV plasmid plasmid.Group 4 (natural types) are accepted saline injection.When the 0th, the 8th, the 12nd and the 24th week, use plasmid with the dosage of 1.5mg/ construct/immunity.Intramuscular injection through behind the EP in vivo comes monkey is carried out immunity.

The DNA plasmid of use electroporation is sent and is induced intensive, the immunoreation of height proliferative cell.At first, estimate inducing of cell immune response in each animal through IFN γ ELISpot.Analysis produces at the SIV specificity IFN γ of each immunity back two all isolating PBMC.(be respectively 60 ± 49 and 569 ± 248SFU/10 in inoculation for the first time ⁶PBMC) the back combined immunization of comparing plasmid rhIL-12 with independent DNA causes nine times of increases of ELISpot number.This early stage enhancing reduces along with further immunity.In four immunity each time after, observe the enhancing of cell response, finally in the anti-SIV IFN of intensive γ ELISpot, reach average out to 16,000SFU/10 ⁶PBMC or 1.6% PBMC colony (Fig. 1 a-1e).DNA+RANTES group shows consistent low ELISpot reaction, especially after immunity for the third time, organizes with DNA+12 (p=0.001) when they are markedly inferior to DNA (p < 0.001).Four times immunity back DNA+RANTES group has 9,217 ± 2111SFU10 ⁶Total IFN gamma reaction of PBMC.

The check of the SIVpol-specificity IFN gamma reaction after the 4th immunity discloses three groups and all has the whole immunogenic big width reaction of covering (table 4).Suppose that an epi-position can appear at based in 3 peptides of 11 amino acid whose eclipsed potentially, then the epi-position number will be approximately 1/3rd of the peptide sum that occurs in the positive storehouse.Ironically, although the DNA+RANTES group has the size that is lower than other inoculation group, has the most different reaction that 68 ± 3.67 epi-positions are estimated in identification.

Then, use the CFSE proliferation assay to come vaccine evaluation inductive CD4 ⁺And CD8 ⁺(Fig. 2 a) for the multiplication capacity of t cell responses.The CD4 that the DNA group has the DNA+12 of being higher than and DNA+RANTES group (being respectively 4.56 ± 2.72% and 1.73 ± 0.76% and 0.46 ± 0.39%, Fig. 2 b) ⁺The T cell proliferative response.Vaccine-induced CD8 ⁺T cell and CD4 ⁺The T cellular compartment is compared has higher multiplication capacity.With CD4 ⁺T cell responses is the same, and 18.7 ± 5.02% in DNA group and the DNA+12 group compared with 17.9 ± 5.33% during DNA+RANTES organizes, and has cell proliferation and be 41.3 ± 9.2% highest response (Fig. 2 c).Though the DNA group has higher proliferative response, difference is not remarkable on statistics.

The combined immunization of plasmid rhIL-12 is through antigen-specific CD4 ⁺The T cell improves cytokine production for measuring the quality of vaccine-induced reaction, estimates SIVpol specific C D4 ⁺The T cell produces IFN γ, IL-2, TNF α and CD107a, and (Fig. 3 a).Use boolean to establish door (Booleangating) and estimate the individual cells generation cell multiplex factor, that is, and vaccine-induced CD4 ⁺The polyfunctional ability of t cell responses.Immunity with independent DNA causes 0.52 ± 0.29% average SIVpol reaction (Fig. 3 b).IL-12 is added immunity cause CD4 ⁺The increase (0.97 ± 0.26%) of T functional response almost twice.Ironically, the reaction (being respectively 0.07 ± .03% and 0.12 ± 0.12%) that is higher than control group is not induced in the interpolation of RANTES.Though the major part reaction in whole inoculation group is for single functional, the DNA+12 group has the cell that can have two kinds or more kinds of functions of maximum ratio, and main difunctional colony is for producing IFN γ ⁺TNF α ⁺Cell.

The combined immunization of plasmid rhIL-12 helps through CD8 ⁺The T cell produces IFN γ at CD4 ⁺Find out obvious phenotypes difference in the T cellular compartment, estimated CD8 then ⁺The phenotype of T cellular compartment.SIVpol specific C D8 upchecks ⁺The cytokine that the T cell produces.The size of functional response has reflected at CD4 ⁺Viewed result in the T cellular compartment.The DNA+12 group has highest response, has 2.34 ± 0.62% CD8 ⁺The T cell produces at least a function (Fig. 3 c).The DNA group has lower slightly reaction, has 1.69 ± 0.63%, and the DNA+RANTES group has low four times reaction, has 0.44 ± 0.16% CD8 ⁺T cell responses.

Except reaction with the highest size, compare with an animal in the DNA+RANTES group with two animals in the DNA group, there are four to have the cell colony that can have whole four kinds of functions in six animals in DNA+12 organizes.Although have the more reaction that can have whole four kinds of functions of vast scale, the principal reaction in the DNA+12 group is the single function of an IFN γ colony.This possibly show that IL-12 induces the terminal effector class cell that breaks up of high frequency as the interpolation of molecule adjuvant, and this cell forfeiture produces the ability of the cytokine except IFN γ.Yet, as dye through CD28 and CD95 defined, the memory phenotype of these cells shows that they all are distributed in effect memory T cell and center memory T cell compartment (Fig. 3 d), thereby has supported the functional property of these T cells.

The maintenance of vaccine-induced reaction has been observed significantly inducing of cell immune response through this plasmid vaccine of being sent by EP, experimentizes to confirm whether these colonies can keep in immunoreactive memory stage.Animal was rested eight months and detection cytokine generation in the reaction that the SIVpol that exsomatizes is stimulated.At CD4 ⁺In the T cellular compartment, DNA and DNA+12 group have reduction most the SIVpol reaction (being respectively 0.15 ± 0.11% and 0.42 ± 0.09%) of the half the or littler level of the immunoreation size after the immunity (Fig. 4 a).An exception is the RANTES group with 0.47 ± 0.37% average response.Yet an animal in group has unusual high IFN gamma reaction, the anamnestic response that this explanation is big.As the 4th immunity back was viewed, the single functioning cell of IFN γ was the main colony in all organizing.

Observe at memory CD8 ⁺Immunoreactive identical reduction (Fig. 4 b) in the T cellular compartment.Though do not detect four kinds of functioning cells, whole three CD107a that the group maintenance can detect ⁺IFN γ ⁺TNF alpha reaction and after the 4th immunity, observe big IFN γ ⁺List function colony.These data show that the enhanced DNA inoculation that has or do not have molecule adjuvant can induce secular multipurpose memory reaction.

Cause in the remarkable decline of a setting point virus load next experimentizing with the combined immunization of plasmid IL-12 and RANTES to confirm after the SIVmac251 mucous membrane excites, whether can influence virus load in these vaccine specific cell responses that kept 8 months after the immunity.SIVmac251 with 25 monkey infective doses (MID) excites animal through the internal rectum approach.In two months weekly and subsequent estimate virus load every month up to exciting back 9 months.Be accumulated at together, inoculation animal as a whole (no matter IL-12 or RANTES) shows that (Fig. 5 a) with respect to the significantly lower peak viremia (p=0.027) of nonvaccinated contrast and lower a setting point (p=0.01).Disclose virus replication control enhancing (Fig. 5 b) in time in the DNA+RANTES group for each further check of organizing average virus load.When the peak of viremia, DNA and DNA+12 group is compared virus load with reduction (be respectively 1.12 and 0.82log) with natural group.RANTES is added immunity cause the 2.0log reduction (p=0.016) significantly of peak viremia.At a setting point, compare the virus load reduction that the DNA group on average only shows 0.62log with natural group.DNA+12 compares the reduction at a setting point virus load with significant 1.5 (p=0.029) and 2.2 (p=0.003) log with the DNA+RANTES group with natural group.Yet, last in this research, when the assay determination through TG-AUC, only the DNA+RANTES group has the remarkable reduction (p=0.008) (Fig. 5 c) of virus replication.

SIV before excites research to identify the some MHC-I allelotrope relevant with the natural control of virus replication.In order to estimate the potential contribution of host MHC-I heredity in this research, carry out the somatotype (table 5) based on comprehensive sequence of all animals conversely.Ex-post analysis discloses has four to express the allelic animals of protectiveness Mamu-B*003, in the DNA+RANTES group, have two and in DNA and DNA+12 group, respectively have one.We also identify the Mamu-B*017 animal in the DNA+12 group.Discovery other animal in the DNA+RANTES group has the new I class allelotrope that is similar to Mamu-B*01702.Observe significantly low virus load (p=0.033) (Fig. 5 d) in the animal of protected monomer type though have at last in this research, (p=0.968) and a setting point (p=0.161) virus load does not have significant difference at the peak.Therefore as if, these protectiveness allelotrope can help to control virus replication between long-term period of infection, but they are not relevant to the influence of virus replication control with the early stage observed vaccine in infection back.

The DNA CD4 of SIVmac251 mucous membrane after exciting that inoculate against ⁺The a setting point place of T cell depletion in inoculation group observed the reduction of virus replication level, estimates protection through other immune parameter.For this reason, per two weeks of 2 months and every thereafter CD4 that monitors all around ⁺The T cell counting.The CD4 that takes place to continue in natural group of after exciting nine month ⁺The T cell depletion is compared with reference count and to be caused 50% loss (Fig. 6 a-6e).On the contrary, inoculation group does not show CD4 ⁺The Cytometric any lasting reduction of T.Therefore the DNA inoculation that has or do not have immunological adjuvant prevents to infect the animal CD4 in back nine months with SIVmac251 ⁺The T cell depletion.

Behind the RANTES combined immunization at the antigen-specific CD8 of peripheral blood ⁺The enhancing that does not have CCR5 to express on the T cell.Experimentize to confirm whether can influence the antigen-specific CD8 that can be mobilized sites of infection and after exciting, influence virus replication with the combined immunization of RANTES ⁺The level of T cell.CD107a, IL-2, IFNg and TNFa on the cell of the immunity isolating freezing preservation in back for the third time carry out ICS.At first measure the SIVpol specific reaction and measure the CCR5 expression on those cells for each function.DNA+RANTES group (0.26 ± 0.09%) in periphery is compared the antigen-specific CD8 that expresses CCR5 with DNA with DNA+12 group (being respectively 0.62 ± 0.28% and 0.33 ± 0.14%) ⁺The frequency of T cell is lower, and (Fig. 7 a).Yet this difference be not on the statistics significantly (Kruskal-Wallis, p=0.056).At CD4 ⁺See similar trend in the T cellular compartment, the DNA group has SIVpol specific C CR5 ⁺Highest frequency of cell (0.27 ± 0.09%) and DNA+RANTES have low-limit frequency (0.13 ± 0.04%) (Fig. 7 b) in peripheral blood.These data support RANTES to regulate or the expression of CCR5 on T cells with antigenic specificity perhaps alternatively mobilizes these to arrive other immune site.

Discuss

Reported in the exploratory study of primate that before this EP and plasmid IL-12 significantly strengthen the ability of HIV plasmid immunogenicity of antigens.In mice study, also observing RANTES is very effective adjuvant.Experimental design as herein described is used for confirming whether these strategies can strengthen the immunogenicity of plasmid SIV vaccine and characterize these reactions have the cell response that helps virus replication control in the hope of evaluation any subpopulation.Test and the total SIV antigen of check with confirm the enhanced reaction whether can influence coupling not, mucous membrane SIVmac251 virus excites.

Consistent with previous work, EP can strengthen vaccine specific IFN gamma reaction to very high level significantly.Opposite with early stage research, the size of reaction surpasses more than 3 times in this research, this with add this research in the optimisation strategy of construct of use consistent.Yet,, further do not strengthen when plasmid IL-12 is observed the ELISpot counting during as adjuvant except this research early stage.This possibly be because DNA dosage or the DNA concentration used are higher; Compare with the 2mg/mL in the exploratory study, with 10mg/mL preparation vaccine.Use the DNA construct of height optimization as if to overcome with higher dosage and concentration when the vaccine of preparation observed immunogenicity difference during with dose delivery lower, that more dilute.This type of preparation possibly have material impact to the DNA platform.

Though observe significant IFN γ ELISpot counting, importantly this parameter should not be separately unique factor of confirming that vaccine candidate object is renderd a service, this is that reaction directly is not associated with the control of virus replication because many researchs have shown ELISpot.For this reason, estimated and confirmed the many parameters required effective HIV vaccine.At first, the result who derives from up-to-date Merck STEP has shown that wide immunoreactive to induce for successful vaccine be important.On average, have with the experimenter of Merck reorganization Ad5 carrier inoculation and be confined to three reaction width to five epi-positions.When estimating through the matrix peptide mapping to the antigenic reaction of SIVpol, observe unprecedented positive reaction for most of peptides storehouse, this is illustrated in the wide epi-position coverage in whole immune group.What also have the meaning is to induce the DNA+RANTES vaccine of most different epi-position reactions to have best virus replication control.

Research among the long-term non-progress person has shown that non-progress person has than progress person's more function property CD8 ⁺The T cell, this shows that these multi-functional colonies possibly be important in virus control.In two groups of DNA and DNA+12, for CD4 ⁺And CD8 ⁺Both observe inducing of polyfunctional reactant the T cellular compartment.Yet interesting observations is very few.At first, at CD4 ⁺In the T cellular compartment, the reactivity to SIVpol in the DNA group is lower than the DNA+12 group with the reaction size.Secondly, at CD4 ⁺And CD8 ⁺The T cellular compartment is among both, and the DNA+12 group has a high proportion of its reaction of being made up of the single functioning cell of IFN γ.In T cell memory differentiation model, the single functioning cell of this type of IFN γ is considered to represent the end effect thing.In order to tackle this possibility, the memory phenotype that dyes and check these single functioning cells through CD28 and CD95.Under this situation, find that these reactions are by from CD28 ⁺CD95 ⁺Center memory colony and CD28 ^-CD95 ⁺Both cells of effector colony are formed.Might CD28 ⁺CD95 ⁺In fact cell is not unifunctional, and has NE some other function in our the multi-functional group.This hypothesis merits attention in research in the future.

Many dna primers/virus vector strengthens research and has been presented at some effectiveness that SHIV89.6P excites virus load reduction in the model.Yet the practicality of this model in the prediction vaccine potency is also disputable.Therefore estimated at stricter, high dosage SIVmac251 mucous membrane and excited the immunoreactive effectiveness of dna vaccination inductive in the model.Reported the influence of using this other research that excites model virus replication to be had appropriateness.

There is significant difference all observing between inoculation animals and the control animal in the loss of protection cd4 t cell.For the whole process of research, prevented the cd4 t cell loss of inoculation animal, and irrelevant with adjuvant.In addition, after exciting, see the difference in virus replication control between the inoculation group.Though DNA and DNA+12 organize both reductions with respect to the about 1log of nonvaccinated contrast demonstration peak viremia, the DNA+RANTES group is compared the reduction of the peak virus load that shows significant 2.2log with natural group.Virus load reduces though the DNA group does not have significantly at a setting point, and the group of assisting a ruler in governing a country with IL-12 and RANTES has significantly lower virus load.In the 35th week, compare with natural contrast, only the DNA+RANTES group shows the influence to virus load that continue, significant.

Big quantity research to people and rhesus monkey has shown that some MHC haplotype is relevant with the control naturally of virus.Though research has at first confirmed to come from the MHC-I/ disease-related property in the rhesus monkey of India, the increase that the rhesus monkey that will come from China is used for the SIV vaccine research born the responsibility in the demand of the similar research of this research colony with additional completed up to now those researchs.In order to tackle this problem, after exciting research to begin, carry out comprehensive I genoid type somatotype of all animals.Ex-post analysis discloses has four animal expressions to duplicate relevant Mamu-B*003 allelotrope with strong prevention SIV; In DNA+RANTES group, there are two and in DNA and DNA+12 group respectively one.In the DNA+12 group, also identified the Mamu-B*017 animal.Ironically, excite the back in the DNA+RANTES animal that never shows the plasma viral RNA that can detect, to detect the novel allelotrope of protectiveness Mamu-B*017 appearance potentially.Have at last in research and to observe significantly lower virus load (p=0.035) in the allelic animal of these protectiveness, this and this allelic importance of I class is consistent.Yet, by contrast, do not have significant difference at the virus load of peak of protected monomer type animal (p=0.561) and a setting point (p=0.056).Therefore the protected monomer type is relevant with the control of virus between long-term period of infection, but in infecting early stage virus replication control, also observes vaccine effect.In addition, in inoculation group, excite back CD4 ⁺The shortage that the T cell is exhausted is not significantly difference between other animal with the allelic animal of Mamu-B*003 or Mamu-B*017 and inoculation.Summary is got up, and these data show that further DNA inoculation can help the protection of SIVmac251 mucous membrane in exciting.

The mechanism that observed enhanced virus suppresses in the DNA+RANTES group it be unclear that.Can be clear that from our analysis this is not because the size of IFN gamma reaction causes.A kind of hypothesis is that the existence of RANTES between duration of immunity can be regulated CCR5 ⁺CD8 ⁺The frequency of T cell.As to CCR5 ⁺CD4 ⁺That kind that the T cell is reported, these cells revert to sites of infection then, and the CD4 that can target infects ⁺The T cell also provides the result of improvement.In order to tackle this hypothesis, in peripheral blood, measuring the antigen-specific CD8 that expresses CCR5 after the immunity with preliminary mode ⁺The frequency of T cell.In the DNA+RANTES of peripheral blood group, observe the lower frequency of these cells.The lower frequency of this result observed antigen-specific sexual cell in analyzing at other possibly reflect the otherness Regression Model (differential homing pattern) (for example mucous membrane compartment) of the RANTES adjuvant reaction of comparing with other inoculation group.Be clear that in the event in office that RANTES regulates the immunobiology aspect of inductive T cell, and as if this adjusting useful to the host in the situation that virus excites.Need carry out research in the future with this key areas of further exploration.

Though checked many immune parameters, do not had a kind of single analysis can predict the result that virus excites.The control of observed virus replication and uncorrelated with the development of neutralizing antibody in this research, this is because after immunity, do not detect any.Nearest research has shown its cell function, and for example cytotoxicity possibly be the better indicator of protective immunological reaction.Yet, the plain inductive immunoreation of vaccine middle punch is not estimated owing to lack the rhesus monkey specific antibody.As if be clear that based on excitation result, though IFN γ ELISpot reaction is important, from being separation individually other function of the better indicator of virus control.The further research of the immunophenotype of IL-12 and RANTES inductive reaction will be in confirming this research is important in the celelular mechanism of viewed reduction virus replication.

The latest result of Merck STEP test has been stressed many problems that future, T cell class vaccine candidate object need be tackled; Comprise: the serological misgivings of the vaccine carrier that prestores, on the Ad5 platform, significantly improve the requirement of immunoreactive size and width and excite and improve virus control in the model at xenogenesis SIV mucous membrane.In this respect, the dna vaccination method of the combination that the collection data acknowledgement that this paper provides is described herein can be induced with SIV and infected relevant intensive immunoreation, evades simultaneously because the problem that the serology that prestores causes.Possibly meaningfully these vaccines combined with carrier method partly to walk around this serology class problem.In addition, the ability of the size of these reactions and adjusting use immunoreactive direction of molecule adjuvant inductive and phenotype is useful especially instrument for the research of the relevant cognation of vaccine in the SIV model.

Table 3: immune programme for children table

Table 4:SIVpol epitope mapping

Group	Minimum	Maximum
			DNA	56.33±14.76	171±43.11
DNA+12	43.67±12.47	128±37.58
			DNA+RANTES	68±3.67	188±14.12

Maximum number is represented the sum of positive peptide

Minimum through with positive peptide stream (2 or more a plurality of adjacent peptide) divided by 3 and be rounded to nearest integer and calculate.To minimum and the group shown in the maximum epi-position estimated value on average ± S.E.M.

Table 5:MHC-I genotyping

The comprehensive MHC I genoid type that comes from Chinese rhesus monkey is through measuring based on Sanger with based on the somatotype of Roche/454 tetra-sodium order-checking.Use BLASTN to compare from the MHC I class sequence of each animal and the internal database of whole known Mamu sequences.The tetra-sodium order-checking provides obviously bigger overburden depth, but makes that highly similarly the ambiguity between the allelotrope increases because the template of estimating is short.The per-cent of reading sequence consistent with given allelotrope in each animal is>5.0% (Burgundy (burgundy)).Use and control SIV and duplicate animal identification that relevant allelotrope or haplotype (Mamu-B*003 and Mamu-B*017) observe for green.An animal (#4424) is expressed allelic with the highly similar novel Mamu-B of Mamu-B*017, and it also possibly control relevant with SIV.With relevant other allelotrope of control, for example come from India rhesus monkey allelotrope Mamu-A*001, Mamu-B*008 or Mamu-B*047 and come from the Chinese rhesus monkey this group and do not observe.

Embodiment 2

People RANTES (pCCL5 ECRO- ECRO=Ig E, CloseNumeral/ RNA ExcellentChange) synthetic and clone

Design, synthesize and clone the 348bp gene that is used to optimize people CCL5 (pHuCCL5ECRO) in the following manner.Make codon select (codon usage) to be suitable for producing codon bias (the not optimization: 0.81 of the Human genome of high CAI value; Optimize: 0.97).For design and synthetic, with codon be chosen as avoid maybe high (> 80%) or the zone of extremely low (< 30%) GC content.In this respect, confirmed that it is average (57%) that wild-type people CCL5 gene uses rare codon and GC content with high frequency, this can help a spot of fast m RNA turnover (turnover).Therefore, increase GC content (62%) slightly to improve the mRNA transformation period.During optimizing process, avoid following cis acting sequence motif: inner TATA box, chi site and RES, rich AT or the extension of rich GC sequence, ARE, INS, CRS sequential element, Tumor-necrosis factor glycoproteins and RNA secondary structure, (hidden) donor splicing site and acceptor site and branch point (branch point).After the analysis, only identify the negative cis acting motif of 1 negative sense and be removed.The gene of the people CCL5 that optimizes is synthetic through Geneart, and Inc. (Germany) carries out.Assemble the people CCL5 gene that synthetic height codon/RNA optimizes by synthetic oligonucleotide.Introduce Kozak sequence (GCCACC) to strengthen translation initiation, introduce the IgE leader sequence and add two terminator codons to guarantee effective termination.Use EcoRI and XhoI restriction site that fragment cloning is gone into pVAX1.Through VGX medicament (Pure YieldTMplasmid Midiprep, Promega) plasmid DNA purification from the bacterium that transforms, and measure concentration through UV spectrum.Come the final dna sequence dna of confirmer CCL5 (phumCCL5ECRO) through order-checking, and be found to be 100% unanimity.

Embodiment 3

Appended sequence table comprises SEQ ID NO:1-8.SEQ ID NO:1 is the nucleotide sequence of the coding human RANTES of codon/RNA optimization.SEQ ID NO:2 is the aminoacid sequence by the people RANTES of SEQ ID NO:1 coding.SEQ ID NO:3 is that the coding that codon/RAA optimizes has the RANTES nucleic acid sequences to proteins that is connected to the terminal IgE leader sequence of people RANTESN.SEQ ID NO:4 is the RANTES protein with IgE leader sequence of the N-terminal that is connected to the people RANTES that is encoded by SEQ ID NO:3.SEQ ID NO:5 comprises that the coding of optimizing with the disclosed identical codon/RNA of SEQ ID NO:3 has the RANTES nucleic acid sequences to proteins that is connected to the terminal IgE leader sequence of people RANTESN, but has other Kozak sequence at 5 ' non-translational region of construct.SEQ ID NO:6, it is and the identical sequence of SEQ ID NO:4, for having the RANTES protein that is connected to by the IgE leader sequence of the N-terminal of the people RANTES of SEQ ID NO:3 coding.SEQ ID NO:7 comprises that the coding of optimizing with SEQ ID NO:3 and the disclosed identical codon/RNA of SEQ ID NO:5 has the RANTES nucleic acid sequences to proteins of the IgE leader sequence of the N-terminal that is connected to people RANTES; But has other Kozak sequence and in the restriction enzyme sites of 5 ' and 3 ' end at 5 ' non-translational region of the construct of SEQ ID NO:5; Said restriction enzyme sites is useful operation, is included in to insert in the plasmid and clone's construct.SEQ ID NO:8 is the sequence identical with SEQ ID NO:6 with SEQ ID NO:4, is the RANTES protein with IgE leader sequence of the N-terminal that is connected to the people RANTES that is encoded by SEQ ID NO:3 and SEQ ID NO:5.

Claims (29)

1. isolated nucleic acid molecule, it comprises the nucleotide sequence that is selected from by the following group of forming: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQ ID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; With the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide and with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide.

2. isolated nucleic acid molecule according to claim 1, it comprises SEQ ID NO:1.

3. isolated nucleic acid molecule according to claim 1, its 5 ' end that is included in encoding sequence contains the nucleotide sequence of the IgE pilot code sequence that is connected to SEQ ID NO:1.

4. isolated nucleic acid molecule according to claim 1, it comprises SEQ IDNO:3.

5. isolated nucleic acid molecule according to claim 1, its 5 ' end that is included in encoding sequence contain the IgE pilot code sequence that is connected to SEQ ID NO:1 and further comprise the nucleotide sequence of Kozak sequence at 5 ' non-translational region.

6. isolated nucleic acid molecule according to claim 1, it comprises SEQ IDNO:5.

7. expression vector, it comprises the described nucleotide sequence of the claim 1 that is operably connected to the controlling element that in people's cell, acts on.

8. expression vector according to claim 4, wherein said expression vector are plasmid.

9. compsn, it comprises:

A) a plurality of one or more nucleic acid molecule, it comprises one or more nucleotide sequences that are selected from by the following group of forming:

1) be selected from group by following group: by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, with SEQ ID NO:195% homologous nucleotide sequence, with SEQID NO:395% homologous nucleotide sequence, with SEQ ID NO:595% homologous nucleotide sequence, with SEQ ID NO:795% homologous nucleotide sequence; The function fragment that comprises the SEQ ID NO:1 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:3 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:5 of at least 60 Nucleotide; The function fragment that comprises the SEQ ID NO:7 of at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:1 that comprises at least 60 Nucleotide; Function fragment 95% homologous nucleotide sequence with the SEQ ID NO:3 that comprises at least 60 Nucleotide; The nucleotide sequence of the group of forming with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:5 that comprises at least 60 Nucleotide with the function fragment 95% homologous nucleotide sequence of the SEQ ID NO:7 that comprises at least 60 Nucleotide; With

B) one or more immunogenic one or more other nucleotide sequences of encoding.

10. one or more other nucleotide sequences compsn according to claim 9, wherein said b) are on from a plurality of one or more different IPs acid molecules in a) a plurality of nucleic acid molecule described in the part.

11. compsn according to claim 9, wherein a) a plurality of nucleic acid molecule described in the part comprise SEQ ID NO:1.

12. compsn according to claim 9, wherein a) a plurality of nucleic acid molecule described in the part are included in 5 ' of encoding sequence and hold the nucleotide sequence that contains the IgE pilot code sequence that is connected to SEQ ID NO:1.

13. compsn according to claim 9, wherein a plurality of nucleic acid molecule described in the part comprise the nucleotide sequence that contains SEQ ID NO:3.

14. compsn according to claim 9, wherein a) a plurality of nucleic acid molecule described in the part comprise the IgE pilot code sequence that is connected to SEQ ID NO:1 and further comprise the Kozak sequence at 5 ' non-translational region at 5 ' end of encoding sequence.

15. compsn according to claim 9, wherein a) a plurality of nucleic acid molecule described in the part comprise SEQ ID NO:5.

16. compsn according to claim 9, wherein a) and b) part described in nucleotide sequence be operably connected to the controlling element that in people's cell, acts on separately.

17. compsn according to claim 9, wherein a) and b) described in nucleotide sequence be the part of one or more expression vectors.

18. compsn according to claim 17, wherein one or more expression vectors are plasmid.

19. compsn according to claim 9, wherein said immunogen be the relevant antigen of pathogen antigen, cancer or with the relevant antigen of cell of participating in autoimmune disease.

20. compsn according to claim 19, wherein said immunogen are pathogen antigen.

21. regulate immunoreactive method for one kind, it comprises step from the nucleic acid molecule that comprises the described nucleotide sequence of claim 1 to individuality that use.

22. induce the immunogenic immunoreactive method of antagonism for one kind, it comprises step from the described compsn of claim 9 to individuality that use.

23. a recombinant viral vector, it comprises the described nucleotide sequence of claim 1.

24. recombinant viral vector according to claim 23, it further comprises the immunogenic nucleotide sequence of the coding that is operably connected to controlling element.

25. recombinant viral vector according to claim 23, wherein said immunogen be the relevant antigen of pathogen antigen, cancer or with the relevant antigen of cell of participating in autoimmune disease.

26. recombinant viral vector according to claim 25, wherein said immunogen are pathogen antigen.

27. an immunoreactive method of regulating in the individuality, it comprises to said individuality uses the described recombinant viral vector of claim 26.

28. a deactivation pathogenic agent, it comprises the described nucleotide sequence of claim 1.

29. an immune body is with the method for enantiopathy substance, it comprises to said individuality uses the described deactivation pathogenic agent of claim 29.

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